Microarray based multiplex pathogen analysis and uses thereof

ABSTRACT

Provided herein is an internal standard method for determining copy number of a pathogen DNA in an unpurified nucleic acid sample by using a known copy number of synthetic DNA that shares a consensus region sequence with the pathogen. The sample is subject to two amplification steps using locus-specific primers and fluorescent primers respectively to obtain fluorescent amplicons for the pathogen and synthetic DNA. These are hybridized with immobilized pathogen-specific and synthetic DNA-specific nucleic acid probes and imaged to obtain fluorescent signals for pathogen-specific and synthetic DNA-specific amplicons. Signal intensities are correlated with the known copy number of synthetic DNA to determine copy number of pathogen DNA in the plant. Also described herein is a method to simultaneously quantitate using the above method, copy numbers of both pathogen and plant DNA in a sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional under 35 U.S.C. § 120 of pendingapplication U.S. Ser. No. 16/158,276, filed Oct. 11, 2018, which is acontinuation-in-part under 35 U.S.C. § 120 of pending application U.S.Ser. No. 15/916,036, filed Mar. 8, 2018, which is a continuation-In-partunder 35 U.S.C. § 120 of pending non-provisional application U.S. Ser.No. 15/388,561, filed Dec. 22, 2016, which claims benefit of priorityunder 35 U.S.C. § 119(e) of provisional application U.S. Ser. No.62/271,371, filed Dec. 28, 2015, all of which are hereby incorporated intheir entireties.

BACKGROUND OF THE INVENTION Field of the Invention

The present disclosure is in the technical field of DNA based pathogenand plant analysis. More particularly, the present disclosure is in thetechnical field of pathogen analysis for plant, agriculture, food andwater material using a multiplex assay and a 3-dimensional latticemicroarray technology for immobilizing nucleic acid probes.

Description of the Related Art

Current techniques used to identify microbial pathogens rely uponestablished clinical microbiology monitoring. Pathogen identification isconducted using standard culture and susceptibility tests. These testsrequire a substantial investment of time, effort, cost as well as labileproducts. Current techniques are not ideal for testing large numberssamples. Culture-based testing is fraught with inaccuracies whichinclude both false positives and false negatives, as well as unreliablequantification of colony forming units (CFUs). There are issues with thepresence of viable but non-culturable microorganisms which do not showup using conventional culture methods. Certain culture tests arenon-specific in terms of detecting both harmful and harmless specieswhich diminishes the utility of the test to determine if there is athreat present in the sample being tested.

In response to challenges including false positives and culturing ofmicroorganisms, DNA-based diagnostic methods such as polymerase chainreaction (PCR) amplification techniques were developed. To analyze apathogen using PCR, DNA is extracted from a material prior to analysis,which is a time-consuming and costly step.

In an attempt to eliminate the pre-analysis extraction step of PCR,Colony PCR was developed. Using cells directly from colonies from platesor liquid cultures, Colony PCR allows PCR of bacterial cells withoutsample preparation. This technique was a partial success but was not assensitive as culture indicating a possible issue with interference ofthe PCR by constituents in the specimens. Although this possibleinterference may not be significant enough to invalidate the utility ofthe testing performed, such interference can be significant for highlysensitive detection of pathogens for certain types of tests.Consequently, Colony PCR did not eliminate the pre-analysis extractionstep for use of PCR, especially for highly sensitive detection ofpathogens.

It is known that 16s DNA in bacteria and the internal transcribed spacer2 (ITS2) locus in yeast or mold DNA can be PCR amplified, and onceamplified can be analyzed to provide information about the specificbacteria or specific mold or yeast contamination in or on plantmaterial. Further, for certain samples such as blood, fecal matter andothers, PCR may be performed on the DNA in such samples absent anyextraction of the DNA. However, for blood it is known that the result ofsuch direct PCR is prone to substantial sample to sample variation dueto inhibition by blood analytes. Additionally, attempts to performdirect PCR analysis on plant matter have generally been unsuccessful,due to heavy inhibition of PCR by plant constituents.

Over time, additional methods and techniques were developed to improveon the challenges of timely and specific detection and identification ofpathogens. Immuno-assay techniques provide specific analysis. However,the technique is costly in the use of chemical consumables and has along response time. Optical sensor technologies produce fast real-timedetection but such sensor lack identification specificity as they offera generic detection capability as the pathogen is usually opticallysimilar to its benign background. Quantitative Polymerase Chain Reaction(qPCR) technique is capable of amplification and detection of a DNAsample in less than an hour. However, qPCR is largely limited to theanalysis of a single pathogen. Consequently, if many pathogens are to beanalyzed concurrently, as is the case with plant, agriculture, food andwater material, a relatively large number of individual tests areperformed in parallel.

Biological microarrays have become a key mechanism in a wide range oftools used to detect and analyze DNA. Microarray-based detectioncombines DNA amplification with the broad screening capability ofmicroarray technology. This results in a specific detection and improvedrate of process. DNA microarrays can be fabricated with the capacity tointerrogate, by hybridization, certain segments of the DNA in bacteriaand eukaryotic cells such as yeast and mold. However, processing a largenumber of PCR reactions for downstream microarray applications is costlyand requires highly skilled individuals with complex organizationalsupport. Because of these challenges, microarray techniques have not ledto the development of downstream applications.

It is well known that DNA may be linked to a solid support for thepurposes of DNA analysis. In those instances, the surface-associated DNAis generally referred to as the “Oligonucleotide probe” (nucleic acidprobe, DNA probe) and its cognate partner to which the probe is designedto bind is referred to as the Hybridization Target (DNA Target). In sucha device, detection and-or quantitation of the DNA Target is obtained byobserving the binding of the Target to the surface bound Probe viaduplex formation, a process also called “DNA Hybridization”(Hybridization).

Nucleic acid probe linkage to the solid support may be achieved bynon-covalent adsorption of the DNA directly to a surface as occurs whena nucleic acid probe adsorbs to a neutral surface such as cellulose orwhen a nucleic acid probe adsorbs to cationic surface such asamino-silane coated glass or plastic. Direct, non-covalent adsorption ofnucleic acid probes to the support has several limitations. The nucleicacid probe is necessarily placed in direct physical contact with thesurface thereby presenting steric constraints to its binding to a DNATarget as the desired (bound) Target-Probe complex is a double helixwhich can only form by wrapping of the Target DNA strand about the boundProbe DNA: an interaction which is fundamentally inhibited by directphysical adsorption of the nucleic acid probe upon the underlyingsurface.

Nucleic acid probe linkage may also occur via covalent attachment of thenucleic acid probe to a surface. This can be induced by introduction ofa reactive group (such as a primary amine) into the Probe then covalentattachment of the Probe, through the amine, to an amine-reactive moietyplaced upon the surface: such as an epoxy group, or an isocyanate group,to form a secondary amine or a urea linkage, respectively. Since DNA isnot generally reactive with epoxides or isocyanates or other similarstandard nucleophilic substitutions, the DNA Probe must be firstchemically modified with “unnatural” ligands such as primary amines orthiols. While such chemistry may be readily implemented duringoligonucleotide synthesis, it raises the cost of the DNA Probe by morethan a factor of two, due to the cost associated with the modificationchemistry. A related UV crosslinking based approach circumvents the needfor unnatural base chemistry, wherein Probe DNA can be linked to thesurface via direct UV crosslinking of the DNA, mediated by photochemicaladdition of thymine within the Probe DNA to the amine surface to form asecondary amine adduct. However, the need for high energy UV forefficient crosslinking results in substantial side reactions that candamage the nucleic acid probe beyond use. As is the case for adsorptivelinkage, the covalent linkages possible between a modified nucleic acidprobe and a reactive surface are short, in the order of less than 10rotatable bonds, thereby placing the nucleic acid probe within 2 nm ofthe underlying surface. Given that a standard nucleic acid probe is >20bases in length (>10 nm long) a Probe/linker length ratio >10/1 alsoprovides for destabilizing inhibition of the subsequent formation of thedesired Target-Probe Duplex.

Previous Attempts at addressing these problems have not met withsuccess. Attachment of nucleic acid probes to surfaces via theirentrapment into a 3-Dimensional gel phase such as that created bypolymerizing acrylamide and polysaccharides among others have beenproblematic due to the dense nature of the gel phases. While the poresize (about 10 nm) in these gels permit entrapment and/or attachment ofthe nucleic acid probes within the gel, the solution-phase DNA Target,which is typically many times longer than the nucleic acid probe, isblocked from penetrating the gel matrix thereby limiting use of thesegel phase systems due to poor solution-phase access to the Target DNA.Furthermore

Thus, the prior art is deficient in systems and methods for detectingand identifying pathogen DNA, which uses fewer chemical and labileproducts, reduces processing steps and provides faster results whilemaintaining accuracy, specificity and reliability. The prior art is alsodeficient in methods to determine absolute copy numbers ofpathogen-specific DNA in an unpurified nucleic acid sample comprising amultiplicity of pathogens. The present invention fulfills thislong-standing need and desire in the art.

SUMMARY OF THE INVENTION

The present invention is directed to a method for determining theabsolute copy number of DNA for one or more pathogens in a plant tissuesample. The method comprises adding a known copy number of a syntheticDNA as an internal reference standard to an unpurified sample comprisingplant DNA and non-DNA nucleic acids. The synthetic DNA has a centralregion sequence that is distinct from signature sequence determinants inthe pathogen DNA and 5′ and 3′ end sequences substantially identical toa consensus sequence in the pathogen DNA. A tandem, single assay firstamplification (locus PCR) of both pathogen DNA and the synthetic DNA isperformed, followed by a second amplification (labeling PCR) using thefirst amplification products as template and fluorescent labeled primersto generate fluorescent labeled pathogen-specific and syntheticDNA-specific amplicons. These fluorescent labeled amplicons arehybridized with pathogen DNA and synthetic DNA specific nucleic acidprobes immobilized at known positions on a 3-dimensional latticemicroarray via different fluorescent labeled bifunctional polymerlinkers. A multi-color image of the microarray is obtained, thefluorescent signals superimposed and the sequence of nucleic acid probesat superimposed positions compared with a database of signature sequencedeterminants in pathogens to identify the pathogen. The fluorescentintensities of the hybridized pathogen-specific amplicons is correlatedwith that of the synthetic DNA-specific amplicons and the known copynumber of synthetic DNA added to the sample to determine copy number ofthe pathogen of interest in the sample.

The present invention is also directed to a method for determining theabsolute copy number of DNA for one or more pathogens and plants in aplant tissue sample. The method comprises adding a known copy number ofa synthetic DNA as an internal reference standard to an unpurifiedsample comprising plant DNA and non-DNA nucleic acids. The synthetic DNAhas a central region sequence that is distinct from signature sequencedeterminants in the pathogen DNA and 5′ and 3′ end sequencessubstantially identical to a consensus sequence in the pathogen DNA andplant DNA. A tandem, single assay first amplification (locus PCR) of thepathogen DNA, plant DNA and synthetic DNA is performed, followed by asecond amplification (labeling PCR) using the first amplificationproducts as template and fluorescent labeled primers to generatefluorescent labeled pathogen-specific, plant-specific and syntheticDNA-specific amplicons. These fluorescent labeled amplicons arehybridized with pathogen DNA, plant DNA and synthetic DNA specificnucleic acid probes immobilized at known positions on a 3-dimensionallattice microarray via different fluorescent labeled bifunctionalpolymer linkers. A multi-color image of the microarray is obtained, thefluorescent signals superimposed and the sequence of nucleic acid probesat superimposed positions compared with a database of signature sequencedeterminants in pathogens and plants to identify the pathogen and plantrespectively. The fluorescent intensities of the hybridizedpathogen-specific amplicons and plant-specific amplicons is correlatedwith that of the synthetic DNA-specific amplicons and the known copynumber of synthetic DNA added to the sample to determine copy number ofthe pathogen and plant in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the embodiments ofthe present disclosure will become better understood when the followingdetailed description is read with reference to the accompanying drawingsin which like characters represent like parts throughout the drawing,wherein:

FIGS. 1A-1D illustrate a covalent microarray system comprising probesand bifunctional labels printed on an activated surface. FIG. 1A showsthe components—unmodified nucleic acid probe, amine-functionalized (NH)bifunctional polymer linker and amine-functionalized (NH) fluorescentlylabeled bifunctional polymer linker in a solvent comprising water and ahigh boiling point water-miscible liquid, and a solid support withchemically activatable groups (X). FIG. 1B shows the first step reactionof the bifunctional polymer linker with the chemically activated solidsupport where the bifunctional polymer linker becomes covalentlyattached by the amine groups to the chemically activated groups on thesolid support. FIG. 1C shows the second step of concentration viaevaporation of water from the solvent to increase the concentration ofthe reactants—nucleic acid probes and bifunctional polymer linker. FIG.1D shows the third step of UV crosslinking of the nucleic acid probesvia thymidine base to the bifunctional polymer linker within evaporatedsurface, which in some instances also serves to covalently link adjacentbifunctional polymeric linkers together via crosslinking to the nucleicacid Probe.

FIGS. 2A-2D illustrate an adsorptive microarray system comprising probesand bifunctional polymeric linkers. FIG. 2A shows the components;unmodified nucleic acid probe and functionalized (R_(n)) bifunctionalpolymer linker and similarly functionalized fluorescent labeledbifunctional polymer linker in a solvent comprising water and a highboiling point water-miscible liquid, and a solid support, wherein theR_(n) group is compatible for adsorbing to the solid support surface.FIG. 2B shows the first step adsorption of the bifunctional polymerlinker on the solid support where the bifunctional polymer linkersbecome non-covalently attached by the R_(n) groups to the solid support.FIG. 2C shows the second step of concentration via evaporation of waterfrom the solvent to increase the concentration of the reactants—Nucleicacid probes and bifunctional polymer linker. FIG. 2D shows the thirdstep of UV crosslinking of the nucleic acid probes via thymidine base tothe bifunctional polymer linker and other nucleic acid probes within theevaporated surface which in some instances also serves to covalentlylink adjacent bifunctional polymeric linkers together via crosslinkingto the nucleic acid Probe.

FIGS. 3A-3C show experimental data using the covalent microarray system.In this example of the invention the bifunctional polymeric linker was achemically modified 40 base long oligo deoxythymidine (OligodT) having aCY5 fluorescent dye attached at its 5′ terminus and an amino groupattached at its 3′ terminus, suitable for covalent linkage with aborosilicate glass solid support which had been chemically activated onits surface with epoxysilane. The nucleic acid probes comprisedunmodified DNA oligonucleotides, suitable to bind to the solution statetarget, each oligonucleotide terminated with about 5 to 7 thymidines, toallow for photochemical crosslinking with the thymidines in the topdomain of the polymeric (oligodT) linker.

FIG. 3A shows an imaged microarray after hybridization and washing, asvisualized at 635 nm. The 635 nm image is derived from signals from the(red) CY5 fluor attached to the 5′ terminus of the bifunctional polymerlinker (OligodT) which had been introduced during microarray fabricationas a positional marker in each microarray spot.

FIG. 3B shows a microarray imaged after hybridization and washing asvisualized at 532 nm. The 532 nm image is derived from signals from the(green) CY3 fluor attached to the 5′ terminus of PCR amplified DNAobtained during PCR Reaction #2 of a DNA containing sample.

FIG. 3C shows an imaged microarray after hybridization and washing asvisualized with both the 532 nm and 635 nm images superimposed. Thesuperimposed images display the utility of parallel attachment of aCY5-labelled OligodT positional marker relative to the sequence specificbinding of the CY3-labelled PCR product.

FIG. 4A is a graphical representation of the position of PCR primersemployed within the 16s locus (all bacteria) to be used to PCR amplifyunpurified bacterial contamination obtained from Cannabis wash andrelated plant wash. These PCR primers are used to amplify and dye labelDNA from such samples for bacterial analysis via microarrayhybridization.

FIG. 4B is a graphical representation of the position of PCR primersemployed within the stx1 locus (pathogenic E. coli) to be used to PCRamplify unpurified bacterial contamination obtained from Cannabis washand related plant wash. These PCR primers are used to amplify and dyelabel DNA from such samples for bacterial analysis via microarrayhybridization.

FIG. 5A is a graphical representation of the position of PCR primersemployed as a two stage PCR reaction within the stx2 locus (pathogenicE. coli) to be used to PCR amplify unpurified bacterial contaminationobtained from Cannabis wash and related plant wash. These PCR primersare used to amplify and dye label DNA from such samples for bacterialanalysis via microarray hybridization.

FIG. 5B is a graphical representation of the position of PCR primersemployed within the invA locus (Salmonella) to be used to PCR amplifyunpurified bacterial contamination obtained from Cannabis wash andrelated plant wash. These PCR primers are used to amplify and dye labelDNA from such samples for bacterial analysis via microarrayhybridization.

FIG. 6 is a graphical representation of the position of PCR primersemployed within the tuf locus (E. coli) to be used to PCR amplifyunpurified bacterial contamination obtained from Cannabis wash andrelated plant wash. These PCR primers are used to amplify and dye labelDNA from such samples for bacterial analysis via microarrayhybridization.

FIG. 7 is a graphical representation of the position of PCR primersemployed within the ITS2 locus (yeast and mold) to be used to PCRamplify unpurified yeast, mold and fungal contamination obtained fromCannabis wash and related plant wash. These PCR primers are used toamplify and dye label DNA from such samples for yeast and mold analysisvia microarray hybridization.

FIG. 8 is a graphical representation of the position of PCR primersemployed within the ITS1 locus (Cannabis Plant Control) to be used toPCR amplify unpurified DNA obtained from Cannabis wash. These PCRprimers are used to amplify and dye label DNA from such samples for DNAanalysis via microarray hybridization. This PCR reaction is used togenerate an internal plant host control signal, via hybridization, to beused to normalize bacterial, yeast, mold and fungal signals obtained bymicroarray analysis on the same microarray.

FIG. 9 is a flow diagram illustrating the processing of unpurifiedCannabis wash or other surface sampling from Cannabis (and related plantmaterial) so as to PCR amplify the raw Cannabis or related plantmaterial, and then to perform microarray analysis on that material so asto analyze the pathogen complement of those plant samples

FIG. 10 is a representative image of the microarray format used toimplement the nucleic acid probes. This representative format comprises12 microarrays printed on a glass slide, each separated by a Teflondivider (left). Each microarray queries the full pathogen detectionpanel in quadruplicate. Also, shown is a blow-up (right) of one suchmicroarray for the analysis of pathogens in Cannabis and related plantmaterials. The Teflon border about each microarray is fit to localizeabout 50 μL fluid sample for hybridization analysis where fluorescentlabeled amplicons and placed onto the microarray for 30 min at roomtemperature, followed by washing at room temperature then microarrayimage scanning of the dye-labelled pathogen and host Cannabis DNA.

FIGS. 11A-11B shows representative microarray hybridization dataobtained from purified bacterial DNA standards (FIG. 11A) and purifiedfungal DNA standards (FIG. 11B). In each case, the purified bacterialDNA is PCR amplified as though it were an unpurified DNA, thenhybridized on the microarray via the microarray probes described above.The data show that in this microarray format, each of the bacteria canbe specifically identified via room temperature hybridization andwashing. Similarly, the purified fungal DNA is PCR amplified as thoughit were an unpurified DNA, then hybridized on the microarray via themicroarray probes described above. The data show that in this microarrayformat, each of the fungal DNAs can be specifically identified via roomtemperature hybridization and washing.

FIG. 12 shows representative microarray hybridization data obtained from5 representative raw Cannabis wash samples. In each case, the rawpathogen complement of these 5 samples is PCR amplified, then hybridizedon the microarray via the microarray probes described above. The datashow that in this microarray format, specific bacterial, yeast, mold andfungal contaminants can be specifically identified via room temperaturehybridization and washing.

FIG. 13 shows representative microarray hybridization data obtained froma representative raw Cannabis wash sample compared to a representative(raw) highly characterized, candida samples. In each case, the rawpathogen complement of each sample is PCR amplified, then hybridized onthe microarray via the microarray probes described above. The data showthat in this microarray format, specific fungal contaminants can bespecifically identified via room temperature hybridization and washingon either raw Cannabis wash or cloned fungal sample.

FIG. 14 shows a graphical representation of the position of PCR primersemployed in a variation of an embodiment for low level detection ofBacteria in the Family Enterobacteriaceae including E. coli. These PCRprimers are used to selectively amplify and dye label DNA from targetedorganisms for analysis via microarray hybridization.

FIG. 15A is a graphical representation of microarray hybridization datademonstrating low level detection of E. coli O157:H7 from certifiedreference material consisting of enumerated colonies of specifiedbacteria spiked onto Humulus lupulus, (Hop plant).

FIG. 15B is a graphical representation of microarray hybridization datademonstrating low level detection of E. coli O1111 from certifiedreference material consisting of enumerated colonies of specifiedbacteria spiked onto Humulus lupulus, (Hop plant).

FIG. 15C is a graphical representation of microarray hybridization datademonstrating low level detection of Salmonella enterica from certifiedreference material consisting of enumerated colonies of specifiedbacteria spiked onto Humulus lupulus, (Hop plant).

FIG. 16 shows diagrams for sample collection and preparation from twomethods. Both the tape pull and wash method are used to process samplesto provide a solution for microbial detection via microarray analysis.

FIG. 17A is a graphical representation of the position of PCR primersused to PCR amplify unpurified bacterial contamination in a Cannabiswash or related plant wash. Two sets of PCR primers are used. The firstset of forward primer (FP) and reverse primer (RP) support a “Locus PCR”step wherein amplification of bacterial recombinant DNA (rDNA) is basedon the 16s locus present in all bacteria. The second set of primerssupport a “Labeling PCR” step wherein the primers are dye labeled andspecific to the bacteria of interest. The two PCR steps in thisinvention differs from FIGS. 4A and 4B in the addition of a known copynumber of a synthetic DNA sequence to the sample, as an internalreference standard, prior to performing the first PCR wherein, thesynthetic DNA sequence is distinguishable from the bacterial sequencesin the sample and have end sequences complementary to the sequence inthe FP and the RP, so that the synthetic DNA sequence is co-amplifiedwith the bacterial sequence in the sample.

FIG. 17B is a graphical representation of the position of PCR primersused to PCR amplify unpurified bacterial contamination in a Cannabiswash or related plant wash. Two sets of PCR primers are used. The firstset of forward primer (FP) and reverse primer (RP) support a “Locus PCR”step wherein amplification of bacterial genomic DNA (DNA) is based onthe 16s locus present in all bacteria. The second set of primers supporta “Labeling PCR” step wherein the primers are dye labeled and specificto the bacteria of interest. The two PCR steps in this invention differsfrom FIGS. 4A and 4B in the addition of a known copy number of asynthetic DNA sequence to the sample, as an internal reference standard,prior to performing the first PCR wherein, the synthetic DNA sequence isdistinguishable from the bacterial sequences in the sample and have endsequences complementary to the sequence in the FP and the RP, so thatthe synthetic DNA sequence is co-amplified with the bacterial sequencein the sample.

FIG. 18 is a graphical representation of the position of PCR primersused to PCR amplify unpurified eukaryotic (e.g. yeast or mold)contamination in a Cannabis wash or related plant wash. Two sets of PCRprimers are used. The first set of forward primer (FP) and reverseprimer (RP) support a “Locus PCR” step wherein amplification of theeukaryotic DNA is based on the ITS2 locus present in all eukaryotes. Thesecond set of primers support a “Labeling PCR” step wherein the primersare dye labeled and specific to the eukaryotes of interest. The two PCRsteps in this invention differs from FIGS. 4A and 4B in the addition ofa known copy number of a synthetic DNA sequence to the sample, as aninternal reference standard, prior to performing the first PCR wherein,the synthetic DNA sequence is distinguishable from the eukaryoticsequences in the sample and have end sequences complementary to thesequence in the FP and the RP, so that the synthetic DNA sequence isco-amplified with the eukaryotic sequence in the sample.

FIG. 19A illustrates calculated copies of ITS2 regions in Aspergillusflavus varied to deliver approximately 0 to 30,000 copies per PCRreaction performed in the presence of 3000 copies (known amount) ofTotal Yeast and Mold Quantitative Control (TYM Quant Control) as theinternal reference standard, using a Total Yeast and Mold nucleic acidprobe for Aspergillus and a TYM Quant Control probe for the internalstandard. The point of crossover at 3000 copies, each for Aspergillusand TYM Quant Control (arrow) between the titration curves forAspergillus (unknown copy number) and the TYM internal referencestandard (known copy number) reveals the copy number of Aspergillus DNAin the sample.

FIG. 19B illustrates calculated copies of ITS2 regions in Saccharomycescerevisiae varied to deliver approximately 0 to 30,000 copies per PCRreaction performed in the presence of 3000 copies (known amount) ofTotal Yeast and Mold Quantitative Control (TYM Quant Control) as theinternal reference standard, using a Total Yeast and Mold nucleic acidprobe for Saccharomyces and a TYM Quant Control probe for the internalstandard. The point of crossover at 3000 copies, each for Saccharomycesand TYM Quant Control (arrow) between the titration curves forSaccharomyces (unknown copy number) and the TYM internal referencestandard (known copy number) reveals the copy number of SaccharomycesDNA in the sample.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of this invention, there is provided a 3-dimensionallattice microarray system for screening a sample for the presence of amultiplicity of DNA. The system comprises a chemically activatable solidsupport, a bifunctional polymer linker and a plurality of nucleic acidprobes designed to identify sequence determinants in plant, animal orpathogen DNA.

In this embodiment, the solid support may be made of any suitablematerial known in the art including but not limited to borosilicateglass, a thermoplastic acrylic resin such aspoly(methylmethacrylate-VSUVT (PMMA-VSUVT), a cycloolefin polymers suchas ZEONOR® 1060R, metals including, but not limited to gold andplatinum, plastics including, but not limited to polyethyleneterephthalate, polycarbonate, nylon, ceramics including, but not limitedto TiO₂, and Indium tin oxide (ITO) and engineered carbon surfacesincluding, but not limited to graphene. The solid support has a frontsurface and a back surface and may be activated on the front surfacewith suitable chemicals which include but are not limited toepoxysilane, isocyanate, succinimide, carbodiimide, aldehyde andmaleimide. These are well known in the art and one of ordinary skill inthis art would be able to readily functionalize any of these supports asdesired. In a preferred embodiment, the solid support is epoxysilanefunctionalized borosilicate glass support.

In this embodiment, the bifunctional polymer linker has a top domain anda bottom end. On the bottom end is attached a first reactive moiety thatallows covalent attachment to the chemically activatable groups in thesolid support. Examples of first reactive moieties include but are notlimited to an amine group, a thiol group and an aldehyde group.Preferably, the first reactive moiety is an amine group. On the topdomain of the bifunctional polymer linker is provided a second reactivemoiety that allows covalent attachment to the oligonucleotide probe.Examples of second reactive moieties include but are not limited tonucleotide bases like thymidine, adenine, guanine, cytidine, uracil andbromodeoxyuridine and amino acid like cysteine, phenylalanine, tyrosineglycine, serine, tryptophan, cystine, methionine, histidine, arginineand lysine. The bifunctional polymer linker may be an oligonucleotidesuch as OligodT, an amino polysaccharide such as chitosan, a polyaminesuch as spermine, spermidine, cadaverine and putrescine, a polyaminoacid, with a lysine or histidine, or any other polymeric compounds withdual functional groups which can be attached to the chemicallyactivatable solid support on the bottom end, and the nucleic acid probeson the top domain. Preferably, the bifunctional polymer linker isOligodT having an amine group at the 5′ end.

In this embodiment, the bifunctional polymer linker may be unmodified.Alternatively, the bifunctional polymer linker has a color orfluorescent label attached covalently. Examples of fluorescent labelsinclude, but are not limited to the fluorescent dyes CY5, DYLIGHT™DY647, ALEXA FLUOR® 647, CY3, DYLIGHT™ DY547, or ALEXA FLUOR® 550. Thesemay be attached to any reactive group including but not limited to,amine, thiol, aldehyde, sugar amido and carboxy on the bifunctionalpolymer linker. The chemistries of such reactive groups are well knownin the art and one or ordinary skill can readily identify a suitablegroup on a selected bifunctional polymer linker for attaching thefluorescent label. Preferably, the bifunctional polymer linker isCY5-labeled OligodT having an amino group attached at its 3′ terminusfor covalent attachment to an activated surface on the solid support.

Also in this embodiment, the present invention provides a plurality ofnucleic acid probes designed with the purpose of identifying sequencedeterminants in plants, animals or pathogens. The nucleic acid probesare synthetic oligonucleotides and have terminal thymidine bases attheir 5′ and 3′ end. The thymidine bases permit covalent attachment ofthe nucleic acid probes to the bifunctional polymer linker by anystandard coupling procedures including but not limited to chemical,photochemical and thermal coupling. Preferably, covalent attachment ofthe nucleic acid probes to the bifunctional polymer linker is byphotochemical means using ultraviolet light.

In this embodiment, the fluorescent label (fluorescent tag) attached tothe bifunctional polymer linker is beneficial since it allows the userto image and detect the position of the individual nucleic acid probes(“spot”) printed on the microarray. By using two different fluorescentlabels, one for the bifunctional polymer linker and the second for theamplicons generated from the DNA being queried, the user can obtain asuperimposed image that allows parallel detection of those nucleic acidprobes which have been hybridized with amplicons. This is advantageoussince it helps in identifying the plant or pathogen comprised in thesample using suitable computer and software, assisted by a databasecorrelating nucleic acid probe sequence and microarray location of thissequence with a known DNA signature in plants, animals or pathogens. Anyemitter/acceptor fluorescent label pairs known in the art may be used.For example, the bifunctional polymer linker may be labeled withemitters such as CY5, DYLIGHT™ DY647, or ALEXA FLUOR® 647, while theamplicons may be labeled with acceptors such as CY3, DYLIGHT™ DY547, orALEXA FLUOR® 550. Preferably, the emitter is CY5 and the acceptor isCY3.

In another embodiment of this invention, there is provided a3-dimensional lattice microarray system for screening a sample for thepresence of a multiplicity of DNA. The system comprises a solid support,a fluorescent labeled bifunctional polymer linker and a plurality ofnucleic acid probes designed to identify sequence determinants in plant,animal or pathogen DNA. In another aspect of this embodiment, there isprovided a 3-dimensional lattice microarray system for quantitativescreening of a sample for copy number of one or more types of DNA. Thesystem comprises a bifunctional polymer linker, a plurality of nucleicacid probes designed to detect copy number of sequence determinants inplant, animal or pathogen DNA and, nucleic acid probes designed todetect copy number of an internal reference standard comprising a knowncopy number of synthetic DNA. The synthetic DNA has a central regionwith a nucleotide sequence distinct from signature sequence determinantsin the unknown DNA being queried, and 5′ and 3′ ends sequencessubstantially identical to a consensus sequence in the unknown DNA. Suchconsensus sequences include but are not limited to the sequences shownin SEQ ID NO: 152 and 153. Such a structure for the synthetic DNApermits amplification of the synthetic DNA by the same pair of PCRprimers used to amplify the hypervariable region of the unknown DNAbeing queried. Examples of such synthetic DNA which may be employedinclude but is not limited to the sequences shown in SEQ ID NOS:154-157.

Further in this embodiment, the solid support may be made of anysuitable material known in the art including but not limited toborosilicate glass, a thermoplastic acrylic resin such aspoly(methylmethacrylate-VSUVT (PMMA-VSUVT), a cycloolefin polymers suchas ZEONOR® 1060R, metals including, but not limited to gold andplatinum, plastics including, but not limited to polyethyleneterephthalate, polycarbonate, nylon, ceramics including, but not limitedto TiO₂, and Indium tin oxide (ITO) and engineered carbon surfacesincluding, but not limited to graphene. The solid support has a frontsurface and a back surface and may be activated on the front surfacewith suitable chemicals which include but are not limited toepoxysilane, isocyanate, succinimide, carbodiimide, aldehyde andmaleimide. These are well known in the art and one of ordinary skill inthis art would be able to readily functionalize any of these supports asdesired. In a preferred embodiment, the solid support is epoxysilanefunctionalized borosilicate glass support.

In this embodiment, the bifunctional polymer linker has a top domain anda bottom end. On the bottom end is attached a first reactive moiety thatallows covalent attachment to the chemically activatable groups in thesolid support. Examples of first reactive moieties include but are notlimited to an amine group, a thiol group and an aldehyde group.Preferably, the first reactive moiety is an amine group. On the topdomain of the bifunctional polymer linker is provided a second reactivemoiety that allows covalent attachment to the oligonucleotide probe.Examples of second reactive moieties include but are not limited tonucleotide bases like thymidine, adenine, guanine, cytidine, uracil andbromodeoxyuridine and amino acid like cysteine, phenylalanine, tyrosineglycine, serine, tryptophan, cystine, methionine, histidine, arginineand lysine. The bifunctional polymer linker may be an oligonucleotidesuch as OligodT, an amino polysaccharide such as chitosan, a polyaminesuch as spermine, spermidine, cadaverine and putrescine, a polyaminoacid, with a lysine or histidine, or any other polymeric compounds withdual functional groups which can be attached to the chemicallyactivatable solid support on the bottom end, and the nucleic acid probeson the top domain. Preferably, the bifunctional polymer linker isOligodT having an amine group at the 5′ end.

In one aspect of this embodiment, the bifunctional polymer linker isunmodified. Alternatively, the bifunctional polymer linker has a coloror fluorescent label attached covalently. Examples of fluorescent labelsinclude, but are not limited to the fluorescent dyes CY5, DYLIGHT™DY647, ALEXA FLUOR® 647, CY3, DYLIGHT™ DY547, or ALEXA FLUOR® 550. Thesemay be attached to any reactive group including but not limited to,amine, thiol, aldehyde, sugar amido and carboxy on the bifunctionalpolymer linker. The chemistries of such reactive groups are well knownin the art and one or ordinary skill can readily identify a suitablegroup on a selected bifunctional polymer linker for attaching thefluorescent label. Preferably, the bifunctional polymer linker isCY5-labeled OligodT having an amino group attached at its 3′terminus forcovalent attachment to an activated surface on the solid support.

Also, in this embodiment, the present invention provides a plurality ofnucleic acid probes designed with the purpose of quantitating copynumber of sequence determinants in plants, animals or pathogens and,nucleic acid probes designed to detect an internal reference standardcomprising a known copy number of synthetic DNA. The nucleic acid probesare synthetic oligonucleotides and have terminal thymidine bases attheir 5′ and 3′ end. The thymidine bases permit covalent attachment ofthe nucleic acid probes to the bifunctional polymer linker by anystandard coupling procedures including but not limited to chemical,photochemical and thermal coupling. Preferably, covalent attachment ofthe nucleic acid probes to the bifunctional polymer linker is byphotochemical means using ultraviolet light.

In this embodiment, the fluorescent label (fluorescent tag) attached tothe bifunctional polymer linker is beneficial since it allows the userto image and detect the position of the individual nucleic acid probes(“spot”) printed on the microarray. By using two different fluorescentlabels, one for the bifunctional polymer linker and the second for theamplicons generated from the DNA being queried, the user can obtain asuperimposed image that allows parallel detection of those nucleic acidprobes which have been hybridized with amplicons. Furthermore, by usingtwo different fluorescent labels, one for the bifunctional polymerlinker and the second for the amplicons generated, one can quantitatecopy number of the DNA being queried. This feature is advantageous sinceit allows; identification of the plant or pathogen comprised in thesample using suitable computer and software, assisted by a databasecorrelating nucleic acid probe sequence and microarray location of thissequence with a known DNA signature in plants, animals or pathogens; andin addition, quantification of the copy number of the plant and/orpathogen DNA identified. Any emitter/acceptor fluorescent label pairsknown in the art may be used for imaging and analysis. For example, thebifunctional polymer linker may be labeled with emitters such as CY5,DYLIGHT™ DY647, or ALEXA FLUOR® 647, while the amplicons may be labeledwith acceptors such as CY3, DYLIGHT™ DY547, or ALEXA FLUOR® 550.Preferably, the emitter is CY5 and the acceptor is CY3.

In another embodiment of this invention, there is provided a3-dimensional lattice microarray system for screening a sample for thepresence of a multiplicity of DNA. The system comprises a solid support,a fluorescent labeled bifunctional polymer linker and a plurality ofnucleic acid probes designed to identify sequence determinants in plant,animal or pathogen DNA.

In this embodiment, the solid support has a front surface and a backsurface. The front surface has non-covalent adsorptive properties forspecific functionalized group(s) present in the fluorescent labeledbifunctional polymer linker (described below). Examples of such solidsupport include, but are not limited to borosilicate glass, SiO₂, metalsincluding, but not limited to gold and platinum, plastics including, butnot limited to polyethylene terephthalate, polycarbonate, nylon,ceramics including, but not limited to TiO₂, and Indium tin oxide (ITO)and engineered carbon surfaces including, but not limited to graphene.

In this embodiment, the fluorescent labeled bifunctional polymer linkerhas a top domain and a bottom end. On the bottom end is attached one ormore functional groups (designated by “R_(n)”) that are compatible fornon-covalent adsorptive attachment with the front surface of the solidsupport. Examples of compatible R groups include, but are not limitedto, single stranded nucleic acids (example, OligodT),amine-polysaccharide (example, chitosan), extended planar hydrophobicgroups (example, digoxigenin, pyrene, CY5 dye).

Further in this embodiment, on the top domain of the bifunctionalpolymer linker is provided a second reactive moiety that allows covalentattachment to the oligonucleotide probe. Examples of second reactivemoieties include but are not limited to nucleotide bases like thymidine,adenine, guanine, cytidine, uracil and bromodeoxyuridine and amino acidlike cysteine, phenylalanine, tyrosine glycine, serine, tryptophan,cystine, methionine, histidine, arginine and lysine. To the bottom endof the bifunctional polymer linker may be attached polymeric moleculesincluding, but not limited to an oligonucleotide such as OligodT, anamino polysaccharide such as chitosan, a polyamine such as spermine,spermidine, cadaverine and putrescine, a polyamino acid, with a lysineor histidine, or OligodT that is modified at its 5′ end with adigoxigenin, a pyrene or CY5 or any other polymeric molecules with orwithout chemical modification suitable for non-covalent attachment tothe solid support. On the top domain of these bifunctional polymerlinkers is attached, the nucleic acid probes. Preferably, thebifunctional polymer linker is OligodT.

In one aspect of this embodiment, the bifunctional polymer linker isunmodified. Alternatively, the bifunctional polymer linker may be afluorescent labeled bifunctional polymer linker. The fluorescent labelmay be, but is not limited to the fluorescent dyes CY5, DYLIGHT™ DY647,ALEXA FLUOR® 647, CY3, DYLIGHT™ DY547, or ALEXA FLUOR® 550 attached toany reactive group including but not limited to, amine, thiol, aldehyde,sugar amido and carboxy on the bifunctional polymer linker. Thechemistries of such reactive groups are well known in the art and one orordinary skill can readily identify a suitable group on a selectedbifunctional polymer linker for attaching the fluorescent label.Preferably, the bifunctional polymer linker is CY5-labeled OligodT.

Also, in one aspect of this embodiment, the present invention provides aplurality of nucleic acid probes designed with the purpose ofidentifying sequence determinants in plants, animals or pathogens. Inanother aspect of this embodiment, there is additionally providednucleic acid probes that identify sequence determinants in a syntheticDNA added as an internal reference standard to the unpurified samplebeing queried (see below in the embodiments comprising the claimedmethods) with the purpose of quantitating a DNA copy number for theidentified plants, animals or pathogens. In either embodiment, thenucleic acid probes are synthetic oligonucleotides and have terminalthymidine bases at their 5′ and 3′ end. The thymidine bases permitcovalent attachment of the nucleic acid probes to the bifunctionalpolymer linker by any standard coupling procedures including but notlimited to chemical, photochemical and thermal coupling. Preferably,covalent attachment of the nucleic acid probes to the bifunctionalpolymer linker is by photochemical means using ultraviolet light.

Further in this embodiment, the fluorescent label (fluorescent tag)attached to the bifunctional polymer linker is beneficial since itallows the user to image and detect the position of the individualnucleic acid probes (“spot”) printed on the microarray. By using twodifferent fluorescent labels, one for the bifunctional polymer linkerand the second for the amplicons generated from the DNA being queried,the user can obtain a superimposed image that allows parallel detectionof those nucleic acid probes which have been hybridized with amplicons.This is advantageous since it helps in identifying the plant or pathogencomprised in the sample using suitable computer and software, assistedby a database correlating nucleic acid probe sequence and microarraylocation of this sequence with a known DNA signature in plants, animalsor pathogens. Additionally, by using two different fluorescent labels,one for the bifunctional polymer linker and the second for the ampliconsgenerated from the unknown DNA and the internal reference standard(synthetic DNA, which is added to the sample), one can quantitate copynumber of the DNA being queried. This feature is advantageous since itallows; identification of the plant or pathogen comprised in the sampleusing suitable computer and software, assisted by a database ofsignature sequence determinants for correlating nucleic acid probesequence and microarray location of this sequence to identify theplants, animals or pathogens; and in addition, quantification of thecopy number of the plant and/or pathogen DNA identified.

Any emitter/acceptor fluorescent label pairs known in the art may beused. For example, the bifunctional polymer linker may be labeled withemitters such as CY5, DYLIGHT™ DY647, or ALEXA FLUOR® 647, while theamplicons may be labeled with acceptors such as CY3, DYLIGHT™ DY547, orALEXA FLUOR® 550. Preferably, the emitter is CY5 and the acceptor isCY5.

In yet another embodiment of this invention, there is provided a methodfor fabricating a 3-dimensional lattice microarray system for thepurpose of screening a sample for the presence of a multiplicity of DNAin a sample. The method comprises, contacting a solid support with aformulation comprising a plurality of nucleic acid probes, a pluralityof fluorescent bifunctional polymer linkers and a solvent mixturecomprising water and a high boiling point, water-miscible liquid,allowing a first attachment between the fluorescent bifunctional polymerlinkers and the solid support to proceed, evaporating the water in thesolvent mixture thereby concentrating the nucleic acid probes andfluorescent labeled bifunctional polymer linkers, allowing a secondattachment between the nucleic acid probes and the fluorescentbifunctional polymer linker, and washing the solid support with at leastonce to remove unattached fluorescent bifunctional polymer linkers andnucleic acid probes.

In this embodiment, the contacting step is achieved by standard printingmethods known in the art including, but not limited to piezo-electricprinting, contact printing, ink jet printing and pipetting, which allowa uniform application of the formulation on the activated support. Forthis, any suitable solid support known in the art including but notlimited to borosilicate glass, a polycarbonate, a graphene, a gold, athermoplastic acrylic resin such as poly(methylmethacrylate-VSUVT(PMMA-VSUVT) and a cycloolefin polymer such as ZEONOR® 1060R may beemployed.

In one aspect of this embodiment, the first attachment of thebifunctional polymer linker to the solid support is by non-covalentmeans such as by adsorption or electrostatic binding. In this case, thebifunctional polymer linkers with one or more functional groups(designated by “R_(n)”) on the bottom end, that are compatible forattachment with the front surface of the solid support will be used.Examples of compatible R groups include, but are not limited to, singlestranded nucleic acids (example, OligodT), amine-polysaccharide(example, chitosan), extended planar hydrophobic groups (example,digoxigenin, pyrene, CY5 dye). In another aspect of this embodiment, thefirst attachment of the bifunctional polymer linker to the solid supportis by covalent coupling between chemically activatable groups on thesolid support and a first reactive moiety on the bottom end of thebifunctional polymer linker. Suitable chemicals including but are notlimited to epoxysilane, isocyanate, succinimide, carbodiimide, aldehydeand maleimide may be used for activating the support. These are wellknown in the art and one of ordinary skill in this art would be able toreadily functionalize any of these supports as desired. In a preferredembodiment, a borosilicate glass support that is epoxysilanefunctionalized is used. Examples of first reactive moieties amenable tocovalent first attachment include, but are not limited to an aminegroup, a thiol group and an aldehyde group. Preferably, the firstreactive moiety is an amine group.

In this embodiment, the bifunctional polymer linker has a secondreactive moiety attached at the top domain. Examples of second reactivemoieties include but are not limited to nucleotide bases like thymidine,adenine, guanine, cytidine, uracil and bromodeoxyuridine and amino acidlike cysteine, phenylalanine, tyrosine glycine, serine, tryptophan,cystine, methionine, histidine, arginine and lysine. Preferably, thesecond reactive moiety is thymidine. In this aspect of the invention,the bifunctional polymer linker may be an oligonucleotide such asOligodT, an amino polysaccharide such as chitosan, a polyamine such asspermine, spermidine, cadaverine and putrescine, a polyamino acid, witha lysine or histidine, or any other polymeric compounds with dualfunctional groups which can be attached to the chemically activatablesolid support on the bottom end, and the nucleic acid probes on the topdomain. Preferably, the bifunctional polymer linker is OligodT having anamine group at the 5′ end.

In this embodiment, the bifunctional polymer linkers are modified with afluorescent label. Examples of fluorescent labels include but are notlimited to the fluorescent dyes CY5, DYLIGHT™ DY647, ALEXA FLUOR® 647,CY3, DYLIGHT™ DY547 and ALEXA FLUOR® 550 attached to any reactive groupincluding but not limited to, amine, thiol, aldehyde, sugar amido andcarboxy on the bifunctional polymer linker. The chemistries of suchreactive groups are well known in the art and one or ordinary skill canreadily identify a suitable group on a selected bifunctional polymerlinker for attaching the fluorescent label. Preferably, the bifunctionalpolymer linker used for fabricating the microarray is CY5-labeledOligodT.

The method of fabricating the microarray requires use of a solventmixture comprising water and a water-miscible liquid having a boilingpoint above 100° C. This liquid may be any suitable water-miscibleliquid with a boiling point higher than that of water, so that all thesolvent is not lost during the evaporation step. This allows themolecular reactants—nucleic acid probes and bifunctional linkers to beprogressively concentrated during evaporation. Such controlledevaporation is crucial to the present invention since it controls thevertical spacing between nucleic acid probes their avoiding sterichindrance during the hybridization steps thereby improving accuracy andprecision of the microarray. Examples of high boiling pointwater-miscible solvent include but are not limited to glycerol, DMSO andpropanediol. The ratio or water to high boiling point solvent is keptbetween 10:1 and 100:1 whereby, in the two extremes, upon equilibrium,volume of the fluid phase will reduce due to water evaporation tobetween 1/100th and 1/10^(th) of the original volume, thus giving riseto a 100-fold to 10-fold increase in reactant concentration. In apreferred embodiment, the water-miscible solvent is propanediol and thewater to propanediol ratio is 100:1.

Further in this embodiment, the nucleic acid probes used in the methodof microarray fabrication are designed with terminal thymidine bases attheir 5′ and 3′ end. The thymidine bases permit covalent attachment ofthe nucleic acid probes to the bifunctional polymer linker by anystandard coupling procedures including but not limited to chemical,photochemical and thermal coupling during the fabrication process.Preferably, coupling of the nucleic acid probes to the fluorescentlabeled bifunctional polymer linkers is by photochemical covalentcrosslinking.

In yet another embodiment of this invention, there is provided acustomizable microarray kit. The kit comprises a solid support, aplurality of fluorescent labeled bifunctional polymer linkers, nucleicacid probes and a solvent mixture comprising water and one or more of awater-miscible liquid having a boiling point above 100° C., andinstructions to use the kit. Each of the components comprising this kitmay be individually customized prior to shipping based on the goals ofthe end user.

In this embodiment, the solid support has a front surface and a backsurface and made of any suitable material known in the art including butnot limited to borosilicate glass, a polycarbonate, a graphene, a gold,a thermoplastic acrylic resin such as poly(methylmethacrylate-VSUVT(PMMA-VSUVT) and a cycloolefin polymer such as ZEONOR® 1060R.

In one aspect of this embodiment, the solid support is unmodified andhas properties capable of non-covalent attachment to groups in thebifunctional polymer linker. Alternatively, the solid support isactivated on the front surface with chemically activatable groups whichinclude but are not limited to epoxysilane, isocyanate, succinimide,carbodiimide, aldehyde and maleimide. These are well known in the artand one of ordinary skill in this art would be able to readilyfunctionalize any of these supports as desired. In a preferredembodiment, the solid support is epoxysilane functionalized borosilicateglass support.

In this embodiment, the bifunctional polymer linker has a top domain anda bottom end. In one aspect of this embodiment, to the bottom end of thebifunctional polymer linker are attached one or more functional groups(designated by “R_(n)”), which are compatible for attachment with thefront surface of the solid support in a non-covalent binding. Examplesof such compatible R groups include, but are not limited to, singlestranded nucleic acids (example, OligodT), amine-polysaccharide(example, chitosan), extended planar hydrophobic groups (example,digoxigenin, pyrene, CY5 dye). Alternatively, to the bottom end of thebifunctional polymer linker are attached a first reactive moiety thatallows covalent attachment to chemically activatable groups in the solidsupport. Examples of first reactive moieties include but are not limitedto an amine group, a thiol group and an aldehyde group. Preferably, thefirst reactive moiety is an amine group.

Further in this embodiment, on the top domain of the bifunctionalpolymer linker is provided a second reactive moiety that allows covalentattachment to the oligonucleotide probe. Examples of second reactivemoieties include but are not limited to nucleotide bases like thymidine,adenine, guanine, cytidine, uracil and bromodeoxyuridine and amino acidlike cysteine, phenylalanine, tyrosine glycine, serine, tryptophan,cystine, methionine, histidine, arginine and lysine. The bifunctionalpolymer linker may be an oligonucleotide such as OligodT, an aminopolysaccharide such as chitosan, a polyamine such as spermine,spermidine, cadaverine and putrescine, a polyamino acid, with a lysineor histidine, or any other polymeric compounds with dual functionalgroups for attachment to the solid support from the bottom end, and thenucleic acid probes from the top domain.

In one aspect of this embodiment, the bifunctional polymer linkers aremodified with a fluorescent label. Alternatively, the bifunctionalpolymer linker may be a fluorescent labeled bifunctional polymer linkerwhere the fluorescent label is either of CY5, DYLIGHT™ DY647, ALEXAFLUOR® 647, CY3, DYLIGHT™ DY547, or ALEXA FLUOR® 550 attached to anyreactive group including but not limited to, amine, thiol, aldehyde,sugar amido and carboxy on the bifunctional polymer linker. Thechemistries of such reactive groups are well known in the art and one orordinary skill can readily identify a suitable group on a selectedbifunctional polymer linker for attaching the fluorescent label.Preferably, the bifunctional polymer linker is CY5-labeled OligodT.

Also in this embodiment, the present invention provides a plurality ofnucleic acid probes designed with the purpose of identifying sequencedeterminants in plants, animals or pathogens. The nucleic acid probesare synthetic oligonucleotides and have terminal thymidine bases attheir 5′ and 3′ end. The thymidine bases permit covalent attachment ofthe nucleic acid probes to the bifunctional polymer linker by anystandard coupling procedures including but not limited to chemical,photochemical and thermal coupling. Preferably, covalent attachment ofthe nucleic acid probes to the bifunctional polymer linker is byphotochemical means using ultraviolet light. In an alternative aspect ofthis embodiment, the present invention provides a plurality of nucleicacid probes designed with the purpose of identifying sequencedeterminants in plants, animals or pathogens and, a synthetic DNAinternal reference standard, which is added to the sample to quantitateDNA copy number for the sequence determinants in plants, animals orpathogens. The synthetic DNA has a central region with a nucleotidesequence distinct from signature sequence determinants in the unknownDNA being queried, and 5′ and 3′ ends sequences substantially identicalto a consensus sequence in the unknown DNA. Such consensus sequencesinclude but are not limited to the sequences shown in SEQ ID NO: 152 and153. Such a structure for the synthetic DNA permits amplification of thesynthetic DNA by the same pair of PCR primers used to amplify thehypervariable region of the unknown DNA being queried. Examples of suchsynthetic DNA which may be employed include but is not limited to thesequences shown in SEQ ID NOs: 154-157. In either of these embodiment,the nucleic acid probes are synthetic oligonucleotides and have terminalthymidine bases at their 5′ and 3′ end. The thymidine bases permitcovalent attachment of the nucleic acid probes to the bifunctionalpolymer linker by any standard coupling procedures including but notlimited to chemical, photochemical and thermal coupling. Preferably,covalent attachment of the nucleic acid probes to the bifunctionalpolymer linker is by photochemical means using ultraviolet light.

In yet another embodiment of this invention there is provided a methodfor detecting the presence of one or more pathogens in a plant sample.In this embodiment, the pathogen may be a human pathogen, an animalpathogen or a plant pathogen, such as a bacterium, a fungus, a virus, ayeast, algae or a protozoan or a combination thereof. These pathogensmay be present as constituents of the soil, soilless growth media,hydroponic growth media or water in which the plant sample was grown.The method comprises harvesting the pathogens from the plant sample,isolating total nucleic acids comprising pathogen DNA, performing afirst amplification for generating one or more amplicons from the one ormore pathogens present in the sample in a single, simultaneous step;performing a labeling amplification using as template, the one or moreamplicons generated in the first amplification step to generatefluorescent labeled second amplicons; hybridizing the second ampliconswith the nucleic acid probes immobilized on the fabricatedself-assembled, 3-dimensional lattice microarray described above andimaging the microarray to detect the fluorescent signal, which indicatespresence of the one or more pathogens in a plant sample. In thisembodiment, the pathogens present on the plant surface may be harvestedby washing the plant with water to recover the pathogens, followed byconcentrating by filtration on a sterile 0.4 μm filter. In anotheraspect of this embodiment, pathogens within the plant tissue may beharvested by fluidizing the plant tissue sample and pathogens, followedby centrifuging to get a pellet of plant cells and pathogen cells. Ineither embodiment, the harvested sample is disrupted to release thetotal nucleic acids which is used in the subsequent steps withoutfurther purification.

Also in this embodiment, the sample comprising nucleic acids frompathogens (external pathogens) or nucleic acids from both pathogens andplant (internal pathogens) is used to perform a first amplification ofpathogen DNA using pathogen-specific first primer pairs to obtain one ormore pathogen-specific first amplicons. Any DNA amplificationmethodology, including loop-mediated isothermal amplification (LAMP) orpolymerase chain reaction (PCR) that can selectively amplify the DNAcomplement in the sample may be employed. In a preferred embodiment, theamplification is by PCR. In one embodiment, the pathogen is a bacteriumand the first primer pairs have sequences shown in SEQ ID NOS: 1-12. Inanother embodiment, the pathogen is a fungus and the first primer pairshave sequences shown in SEQ ID NOS: 13-16. An aliquot of first ampliconsso generated is used as template for a second, labelling PCRamplification using fluorescent labeled second primer pairs. The secondprimer pairs are designed to amplify an internal flanking region in theone or more first amplicons to obtain one or more first fluorescentlabeled second amplicons. In one embodiment, the pathogen is a bacteriumand the second primer pairs have sequences shown in SEQ ID NOS: 19-30.In another embodiment, the pathogen is a fungus and the second primerpairs have sequences shown in SEQ ID NOS: 31-34.

Further in this embodiment, the fluorescent labeled second amplicons arehybridized on a 3-dimensional lattice microarray system having aplurality of nucleic acid probes as described in detail above. In thisembodiment, the bifunctional polymer linker has a fluorescent label(that is different from the label on the second amplicon) attachedwhereby, imaging the microarray after hybridization and washing resultsin two distinct fluorescent signals—the signal from the fluorescentbifunctional polymer linker which is covalently linked to the nucleicacid probe during fabrication, which would be detected in each spotcomprised in the microarray, and a second amplicon signal that would bedetected only in those spots where the nucleic acid probe sequence iscomplementary to the second amplicon (originally derived byamplification from the pathogen DNA in the sample). Thus, superimposingthe two images using a computer provides beneficial attributes to thesystem and method claimed in this invention since one can readilyidentify the plant or pathogen comprised in the sample from a databasethat correlates nucleic acid probe sequence and microarray location ofthis sequence with a known DNA signature in plants or pathogens. In apreferred embodiment, the bacterial nucleic acid probes having sequencesshown in SEQ ID NOS: 37-85. and fungal nucleic acid probes havingsequences shown in SEQ ID NOS: 86-125 may be used for this purpose.

Further to this embodiment is a method for detecting plant DNA. Theplant may be a terrestrial plant such as a Humulus or a Cannabis, anaquatic plant, an epiphytic plant or a lithophytic plant that grows insoil, soilless media, hydroponic growth media or water. In a preferredaspect, the plant is a Cannabis. This method comprises the steps ofperforming an amplification on an unpurified complex nucleic acid sampleusing plant-specific first primer pairs to generate plant-specific firstamplicons. In one aspect of this embodiment, the first primer pair hassequences shown in SEQ ID NOS: 17-18. Any DNA amplification methodology,including loop-mediated isothermal amplification (LAMP) or polymerasechain reaction (PCR) that can selectively amplify the DNA complement inthe sample may be employed. Preferably the amplification is by PCR. Thefirst amplicons so generated are used as template for a labelingamplification step using fluorescent labeled second primer pairs thatare designed to amplify an internal flanking region in the one or moreof first amplicons generated in the first amplification step to generateone or more first fluorescent labeled second amplicons. In oneembodiment, the second primer pair has sequences shown in SEQ ID NOS:35-36. The second amplicons are hybridized on a 3-dimensional latticemicroarray system having a plurality of plant-specific nucleic acidprobes, and the microarrays imaged and analyzed as described above foridentifying pathogen DNA. In one aspect of this embodiment, thehybridization nucleic acid probes have sequences shown in SEQ ID NOS:126-128.

In yet another embodiment of this invention, there is provided a methodfor simultaneously detecting resident pathogen DNA and plant DNA in aplant sample in a single assay. In this embodiment, the pathogen may bea human pathogen, an animal pathogen or a plant pathogen, which may be abacterium, a fungus, a virus, a yeast, algae or a protozoan or acombination thereof. These pathogens may be present as constituents ofthe soil, soilless growth media, hydroponic growth media or water inwhich the plant sample was grown. The plant may be a terrestrial plantsuch as a Humulus or a Cannabis, an aquatic plant, an epiphytic plant ora lithophytic plant that grows in soil, soilless media, hydroponicgrowth media or water. Preferably, the plant is a Cannabis.

In this embodiment, the method comprises harvesting a plant tissuesample potentially comprising one or more pathogens, fluidizing theplant tissue sample and the one or more pathogens and isolating totalnucleic acids comprising DNA from at least the plant tissue and DNA fromthe one or more pathogens. In one aspect of this embodiment, the step ofisolating total nucleic acids comprises centrifuging the fluidizedsample to get a pellet of plant cells and pathogen cells which aredisrupted to release the total nucleic acids, which are used in thesubsequent steps without further purification.

Further in this embodiment, a first amplification is performed on theunpurified total nucleic acid sample using one or more of a first primerpair each selective for the one or more pathogen DNA and one or more ofa second primer pair selective for the plant DNA to generate one or morepathogen-specific first amplicons and one or more plant-specific secondamplicons. Any DNA amplification methodology, including loop-mediatedisothermal amplification (LAMP) or polymerase chain reaction (PCR) thatcan selectively amplify the DNA complement in the sample may beemployed. In a preferred embodiment, the amplification is by PCR. In oneembodiment, the pathogen is a bacterium and the first primer pairs havesequences shown in SEQ ID NOS: 1-12. In another embodiment, the pathogenis a fungus and the first primer pairs have sequences shown in SEQ IDNOS: 13-16. In either of these embodiments, the plant-specific secondprimer pairs have sequences shown in SEQ ID NOS: 35-36. An aliquot ofthe first and second amplicons so generated is used as a template for asecond, labeling PCR amplification step using fluorescent labeled thirdprimer pairs having a sequence complementary to an internal flankingregion in the one or more pathogen-specific first amplicons andfluorescent labeled fourth primer pairs having a sequence complementaryto an internal flanking region in the one or more plant-specific secondamplicons. Any DNA amplification methodology, including loop-mediatedisothermal amplification (LAMP) or polymerase chain reaction (PCR) thatcan selectively amplify the DNA complement in the sample may beemployed. In a preferred embodiment, the amplification is by PCR. In oneembodiment, the pathogen is a bacterium and the third primer pairs havesequences shown in SEQ ID NOS: 19-30. In another embodiment, thepathogen is a fungus and the third primer pairs have sequences shown inSEQ ID NOS: 31-34. In either of these embodiments, the plant-specificfourth primer pairs have sequences shown in SEQ ID NOS: 35-36. Thelabeling PCR step results in generation of first fluorescent labeledthird amplicons and second fluorescent labeled fourth ampliconscorresponding to the pathogen and plant DNA respectively in the originalharvested sample. These amplicons are then hybridized on a 3-dimensionallattice microarray system having a plurality of nucleic acid probesspecific to sequence determinants in pathogen DNA or plant DNA.Bacterial nucleic acid probes having sequences shown in SEQ ID NOS:37-85, fungal nucleic acid probes having sequences shown in SEQ ID NOS:86-125 and plant nucleic acid probes having sequences shown in SEQ IDNOS: 126-128. may be used for this purpose. After hybridization,unhybridized amplicons are removed by washing and the microarray imaged.Detection of the first fluorescent labeled third amplicon signalindicates presence of pathogens in the plant sample. Detecting thesecond fluorescent labeled fourth amplicon indicates presence of theplant DNA. Superimposing these two signals with the third fluorescentsignal from the fluorescent bifunctional polymer linker-coupled nucleicacid probes allow simultaneous identification of the pathogen and plantin the sample by correlating nucleic acid probe sequence and microarraylocation of this sequence with a known DNA signature in plants orpathogens. These features provide beneficial attributes to the systemand method claimed in this invention.

In yet another embodiment of this invention there is provided a methodfor both detecting the presence of one or more pathogens andquantitating the copy number of pathogen DNA and plant DNA in a plantsample by introducing a known copy number of a synthetic DNA sequence asan internal reference standard to the plant sample. In this embodiment,the pathogen may be a human pathogen, an animal pathogen or a plantpathogen, such as a bacterium, a fungus, a virus, a yeast, algae or aprotozoan or a combination thereof. These pathogens may be present asconstituents of the soil, soilless growth media, hydroponic growth mediaor water in which the plant sample was grown. The method comprisesharvesting the pathogens from the plant sample, isolating total nucleicacids comprising pathogen DNA, performing a first amplification forgenerating one or more amplicons from the one or more pathogens presentin the sample in a single, simultaneous step; performing a labelingamplification using as template, the one or more amplicons generated inthe first amplification step to generate fluorescent labeled secondamplicons; hybridizing the second amplicons with the nucleic acid probesimmobilized on the fabricated self-assembled, 3-dimensional latticemicroarray described above and imaging the microarray to detect thefluorescent signal, which indicates presence of the one or morepathogens in a plant sample. In this embodiment, the pathogens presenton the plant surface may be harvested by washing the plant with water torecover the pathogens, followed by concentrating by filtration on asterile 0.4 μm filter. In another aspect of this embodiment, pathogenswithin the plant tissue may be harvested by fluidizing the plant tissuesample and pathogens, followed by centrifuging to get a pellet of plantcells and pathogen cells. In either embodiment, the harvested sample isdisrupted to release the total nucleic acids which is used in thesubsequent steps without further purification.

In this embodiment, the synthetic DNA has a central region with anucleotide sequence distinct from signature sequence determinants in theunknown DNA being queried, and 5′ and 3′ ends sequences substantiallyidentical to a consensus sequence in the unknown DNA. Such consensussequences include but are not limited to the sequences shown in SEQ IDNO: 152 and 153. Such a structure for the synthetic DNA permitsamplification of the synthetic DNA by the same pair of PCR primers usedto amplify the hypervariable region of the unknown DNA being queried.Examples of such synthetic DNA which may be employed include but is notlimited to the sequences shown in SEQ ID NOs: 154 (fungus) and 155-157(bacteria).

Also in this embodiment, a known copy number of a synthetic DNA is addedto the sample comprising nucleic acids from pathogens (externalpathogens) or nucleic acids from both pathogens and plant (internalpathogens) and a first amplification is performed usingpathogen-specific first primer pairs to obtain one or morepathogen-specific first amplicons and synthetic DNA specific secondamplicons. Any DNA amplification methodology, including loop-mediatedisothermal amplification (LAMP) or polymerase chain reaction (PCR) thatcan selectively amplify the DNA complement in the sample may beemployed. In a preferred embodiment, the amplification is by PCR. Anysuitable first amplification primer pairs may be used for this purposeand one of skill in this art can easily design these primers based onthe pathogen of interest. In one embodiment, the pathogen is a bacteriumand the first primer pairs have sequences shown in SEQ ID NOS: 1 and 2,or SEQ ID NOS: 3 and 4, or SEQ ID NOS: 5 and 6 or SEQ ID NOS: 7 and 8,or SEQ ID NOS: 9 and 10, or SEQ ID NOS: 11 and 12, or SEQ ID NOS: 137and 138. In another embodiment, the pathogen is a fungus and the firstprimer pairs have sequences shown in SEQ ID NOS: 13 and 14, or SEQ IDNOS: 15 and 16, or SEQ ID NOS: 135 and 136. An aliquot of first andsecond amplicons so generated is used as template for a second,labelling PCR amplification using fluorescent labeled second primerpairs. The second primer pairs are designed to amplify an internalflanking region in the one or more pathogen DNA-specific first ampliconsand the synthetic DNA-specific second amplicons to obtain one or morefirst fluorescent labeled third amplicons and first fluorescent labeledfourth amplicons. Any suitable second amplification primer pairs may beused for this purpose and one of skill in this art can easily designthese primers based on the pathogen of interest. In one embodiment, thepathogen is a bacterium and the second primer pairs have sequences shownin SEQ ID NOS: 19 and 20, or SEQ ID NOS: 21 and 22, or SEQ ID NOS: 23and 24 or SEQ ID NOS: 25 and 26, or SEQ ID NOS: 27 and 28, or SEQ IDNOS: 29 and 30, or SEQ ID NOS: 141 and 30. In another embodiment, thepathogen is a fungus and the second primer pairs have sequences shown inSEQ ID NOS: 31 and 32, or SEQ ID NOS: 33 and 34, or SEQ ID NOS: 139 and140.

Further in this embodiment, the fluorescent labeled second amplicons arehybridized on a 3-dimensional lattice microarray system having aplurality of nucleic acid probes specific to pathogen or synthetic DNAspecific amplicons as described in detail above. Any suitable nucleicacid probes may be used for this purpose and one of skill in this artcan easily design them based on the pathogen of interest. In oneembodiment, the bacterial nucleic acid probes have sequences shown inSEQ ID NOS: 37-85 and the synthetic DNA has sequences shown in SEQ IDNO: 155, SEQ ID NO: 156 corresponding respectively to synthetic DNAspecific nucleic acid probes having sequences shown in SEQ ID NO: 142,SEQ ID NO: 143 and SEQ ID NO: 144. In another embodiment, the fungalnucleic acid probes having sequences shown in SEQ ID NOS: 86-125, thesynthetic DNA has sequences shown in SEQ ID NO: 154 that corresponds tosynthetic DNA specific nucleic acid probes having sequences shown in SEQID NO: 145.

In this embodiment, the bifunctional polymer linker has a fluorescentlabel (that is different from the label on the second amplicon) attachedwhereby, imaging the microarray after hybridization and superimposingthe image results in two fluorescent signals—the signal from thefluorescent bifunctional polymer linker which is covalently linked tothe nucleic acid probe during fabrication, which would be detected ineach spot comprised in the microarray, and the signal from fluorescentlabeled third and fourth amplicons corresponding to pathogen andsynthetic DNA respectively and which would be detected only in thosepositions (spots) where the nucleic acid probe sequence is complementaryto the pathogen specific third amplicon (originally derived byamplification from the pathogen DNA in the sample) and synthetic DNAspecific fourth amplicon. Thus, superimposing the signals using acomputer provides beneficial attributes to the system and method claimedin this invention since one can readily identify the plant or pathogencomprised in the sample from a database that correlates nucleic acidprobe sequence and microarray location of this sequence with a known DNAsignature in plants or pathogens. Further to this embodiment, therelative fluorescence intensities (RFU) from the microarray imagecorresponding to fluorescent pathogen DNA-specific amplicons,fluorescent plant DNA-specific amplicons are analyzed and mathematicallycorrelated with fluorescence intensity for the synthetic DNA-specificamplicons and the known copy number for the synthetic DNA added to thesample, to determine copy numbers of the pathogen DNA and plant DNA inthe sample, the mathematical correlation being;C _(n) /C _(o) =P(S _(n) /S _(o))^(x) where,  Equation #1

C_(n)=the number of microbial DNA copies of each type (n) present in theoriginal sample mixture added to the first of two tandem PCR reactionsused to prepare amplicons for microarray analysis.

C_(o)=the number of known synthetic DNA copies (internal referencestandard) added to the first of two PCR reactions used to prepareamplicons for microarray analysis. C_(o) may be set at any valueincluding but not limited to 100, 500, 3,000 and 5,000 depending on therange of unknown microbial copies which might be encountered. In apreferred embodiment C_(o)=3000.

S_(n)=relative fluorescence units (RFU) signal data obtained after PCRamplification, and microarray hybridization of the nth microbialspecies, followed by image analysis.

S_(o)=relative fluorescence units (RFU) signal data obtained after PCRamplification, and microarray hybridization of the synthetic DNAspecies, followed by image analysis.

X=a complex exponential factor which defines the functional relationshipbetween the Experimental Microarray Data Ratio (S_(n)/S_(o)) to theunderlying ratio of microbial DNA copies vs synthetic DNA standardcopies present in the original sample (C_(n)/C_(o)). X may be a linearfunction or exponential or related functional form or a constant whichis itself a function of amplification parameters and conditions ofmicroarray analysis and imaging. In one aspect, X is an exponentialfactor ranging from about 1 to about 3.

P=A constant which relates the Experimental Microarray Data ratio(S_(n)/S_(o)) to the concentration of amplified PCR product which bindsto the microarray. In one aspect, P may range from about 0.1 to about10.

In yet another embodiment of this invention, there is provided a methodfor simultaneously detecting and quantitating resident pathogen DNA andplant DNA in a plant sample in a single assay by introducing a knowncopy number of a synthetic DNA sequence as an internal referencestandard to the plant sample. In this embodiment, the pathogen may be ahuman pathogen, an animal pathogen or a plant pathogen, which may be abacterium, a fungus, a virus, a yeast, algae or a protozoan or acombination thereof. These pathogens may be present as constituents ofthe soil, soilless growth media, hydroponic growth media or water inwhich the plant sample was grown. The plant may be a terrestrial plantsuch as a Humulus or a Cannabis, an aquatic plant, an epiphytic plant ora lithophytic plant that grows in soil, soilless media, hydroponicgrowth media or water. Preferably, the plant is a Cannabis.

In this embodiment, the method comprises harvesting a plant tissuesample potentially comprising one or more pathogens, fluidizing theplant tissue sample and the one or more pathogens and isolating totalnucleic acids comprising DNA from at least the plant tissue and DNA fromthe one or more pathogens. In one aspect of this embodiment, the step ofisolating total nucleic acids comprises centrifuging the fluidizedsample to get a pellet of plant cells and pathogen cells which aredisrupted to release the total nucleic acids, which are used in thesubsequent steps without further purification. To this unpurified totalnucleic acid sample is added a known copy number of synthetic DNA. Thesynthetic DNA has a central region with a nucleotide sequence distinctfrom signature sequence determinants in the unknown DNA being queried,and 5′ and 3′ ends sequences substantially identical to a consensussequence in the unknown DNA. Such consensus sequences include but arenot limited to the sequences shown in SEQ ID NO: 152 and 153. Such astructure for the synthetic DNA permits amplification of the syntheticDNA by the same pair of PCR primers used to amplify the hypervariableregion of the unknown DNA being queried. Examples of such synthetic DNAwhich may be employed include but is not limited to the sequences shownin SEQ ID NOs: 154 (fungus) and 155-157 (bacteria).

Further in this embodiment, a first amplification is performed on theunpurified total nucleic acid sample using one or more of a first primerpair selective for the pathogen DNA and the synthetic DNA and one ormore of a second primer pair selective for the plant DNA to generate oneor more pathogen-specific first amplicons and one or more plant-specificsecond amplicons and synthetic DNA-specific third amplicons. Any DNAamplification methodology, including loop-mediated isothermalamplification (LAMP) or polymerase chain reaction (PCR) that canselectively amplify the DNA complement in the sample may be employed. Ina preferred embodiment, the amplification is by PCR. Any suitable firstamplification primer pairs may be used for this purpose and one of skillin this art can easily design these primers based on the pathogen andplant of interest. In one embodiment, the pathogen is a bacterium andthe first primer pairs have sequences shown in SEQ ID NOS: 1 and 2, orSEQ ID NOS: 3 and 4, or SEQ ID NOS: 5 and 6 or SEQ ID NOS: 7 and 8, orSEQ ID NOS: 9 and 10, or SEQ ID NOS: 11 and 12, or SEQ ID NOS: 137 and138. In another embodiment, the pathogen is a fungus and the firstprimer pairs have sequences shown in SEQ ID NOS: 13 and 14, or SEQ IDNOS: 15 and 16, or SEQ ID NOS: 135 and 136. In either of theseembodiments, the plant-specific second primer pairs have sequences shownin SEQ ID NOS: 17 and 18. An aliquot of the first, second, and thirdamplicons so generated is used as a template for a second, labeling PCRamplification step using a first fluorescent labeled third primer pairshaving a sequence complementary to an internal flanking region in thefirst amplicons and third amplicons and second fluorescent labeledfourth primer pairs having a sequence complementary to an internalflanking region in the one or more plant-specific second amplicons toobtain pathogen DNA-specific first fluorescent labeled fourth amplicons,plant DNA-specific second fluorescent labeled fifth amplicons andsynthetic DNA-specific first fluorescent labeled sixth amplicons. AnyDNA amplification methodology, including loop-mediated isothermalamplification (LAMP) or polymerase chain reaction (PCR) that canselectively amplify the DNA complement in the sample may be employed. Ina preferred embodiment, the amplification is by PCR. Any suitable secondamplification primer pairs may be used for this purpose and one of skillin this art can easily design these primers based on the pathogen andplant of interest. In one embodiment, the pathogen is a bacterium andthe second primer pairs have sequences shown in SEQ ID NOS: 19 and 20,or SEQ ID NOS: 21 and 22, or SEQ ID NOS: 23 and 24 or SEQ ID NOS: 25 and26, or SEQ ID NOS: 27 and 28, or SEQ ID NOS: 29 and 30, or SEQ ID NOS:141 and 30. In another embodiment, the pathogen is a fungus and thesecond primer pairs have sequences shown in SEQ ID NOS: 31 and 32, orSEQ ID NOS: 33 and 34, or SEQ ID NOS: 139 and 140. In either of theseembodiments, the plant-specific fourth primer pairs have sequences shownin SEQ ID NOS: 35-36.

Further in this embodiment, the fourth, fifth and sixth amplicons arethen hybridized on a 3-dimensional lattice microarray system having aplurality of nucleic acid probes specific to sequence determinants inpathogen DNA, plant DNA or synthetic DNA. Any suitable nucleic acidprobes may be used for this purpose and one of skill in this art caneasily design them based on the pathogen of interest. In one embodiment,the bacterial nucleic acid probes have sequences shown in SEQ ID NOS:37-85 and the synthetic DNA has sequences shown in SEQ ID NO: 155, SEQID NO: 156 corresponding respectively to synthetic DNA specific nucleicacid probes having sequences shown in SEQ ID NO: 142, SEQ ID NO: 143 andSEQ ID NO: 144. In another embodiment, the fungal nucleic acid probeshaving sequences shown in SEQ ID NOS: 86-125, the synthetic DNA hassequences shown in SEQ ID NO: 154 that corresponds to synthetic DNAspecific nucleic acid probes having sequences shown in SEQ ID NO: 145.In either embodiment, plant nucleic acid probes having sequences shownin SEQ ID NOS: 126-128. In this embodiment, the nucleic acid probes areattached to the microarray via a third fluorescent label bifunctionalpolymer linker has a (third fluorescent label is different from thefirst and second fluorescent label on the amplicons). Thereby, imagingof the hybridized amplicons on the microarray gives fluorescentsignals—the third fluorescent signal from the nucleic acid probes thatare attached to the bifunctional polymer linker, first fluorescentsignal from the hybridized pathogen-specific fourth, and syntheticDNA-specific sixth amplicons and second fluorescent signal from thehybridized plant-specific fifth amplicons. Superimposing each of thefirst and second fluorescent third signals with the third fluorescentsignal from the nucleic acid probe using a computer provides beneficialattributes to the system and method claimed in this invention since onecan readily identify the plant or pathogen comprised in the sample froma database that correlates nucleic acid probe sequence and microarraylocation of this sequence with a known DNA signature in plants orpathogens. Further to this embodiment, the relative fluorescenceintensities (RFU) from the microarray image corresponding to fluorescentpathogen DNA-specific amplicons, fluorescent plant DNA-specificamplicons are analyzed and mathematically correlated with fluorescenceintensity for the synthetic DNA-specific amplicons and the known copynumber for the synthetic DNA added to the sample, to determine copynumbers of the pathogen DNA and plant DNA in the sample, themathematical correlation being;C _(n) /C _(o) =P(S _(n) /S _(o))^(x) where,  Equation #1

C_(n)=the number of microbial DNA copies of each type (n) present in theoriginal sample mixture added to the first of two tandem PCR reactionsused to prepare amplicons for microarray analysis.

C_(o)=the number of known synthetic DNA copies (internal referencestandard) added to the first of two PCR reactions used to prepareamplicons for microarray analysis. C_(o) may be set at any valueincluding but not limited to 100, 500, 3,000 and 5,000 depending on therange of unknown microbial copies which might be encountered. In apreferred embodiment C_(o)=3000.

S_(n)=relative fluorescence units (RFU) signal data obtained after PCRamplification, and microarray hybridization of the nth microbialspecies, followed by image analysis.

S_(o)=relative fluorescence units (RFU) signal data obtained after PCRamplification, and microarray hybridization of the synthetic DNAspecies, followed by image analysis.

X=a complex exponential factor which defines the functional relationshipbetween the Experimental Microarray Data Ratio (S_(n)/S_(o)) to theunderlying ratio of microbial DNA copies vs synthetic DNA standardcopies present in the original sample (C_(n)/C_(o)). X may be a linearfunction or exponential or related functional form or a constant whichis itself a function of amplification parameters and conditions ofmicroarray analysis and imaging. In one aspect, X is an exponentialfactor ranging from about 1 to about 3.

P=A constant which relates the Experimental Microarray Data ratio(S_(n)/S_(o)) to the concentration of amplified PCR product which bindsto the microarray. In one aspect, P may range from about 0.1 to about10.

In yet another embodiment of the present disclosure there is provided amethod for DNA based pathogen analysis. The embodiments of the presentdisclosure use DNA amplification methodologies, including loop-mediatedisothermal amplification (LAMP) or polymerase chain reaction (PCR) teststhat can selectively amplify the DNA complement of that plant materialusing unpurified plant and pathogen material. The embodiments are alsobased on the use of aforementioned PCR-amplified DNA as the substratefor microarray-based hybridization analysis, wherein the hybridizationis made simple because the nucleic acid probes used to interrogate theDNA of such pathogens are optimized to function at room temperature.This enables the use of the above-mentioned microarray test at ambienttemperature, thus bypassing the prior art requirement that testing besupported by an exogenous temperature-regulating device.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

Example 1 Fabrication of 3-Dimensional Lattice Microarray Systems

The present invention teaches a way to link a nucleic acid probe to asolid support surface via the use of a bifunctional polymeric linker.The nucleic acid probe can be a PCR amplicon, syntheticoligonucleotides, isothermal amplification products, plasmids or genomicDNA fragment in a single stranded or double stranded form. The inventioncan be sub-divided into two classes, based on the nature of theunderlying surface to which the nucleic acid probe would be linked.

Covalent Microarray System with Activated Solid Support

The covalent attachment of any one of these nucleic acid probes does notoccur to the underlying surface directly, but is instead mediatedthrough a relatively long, bi-functional polymeric linker that iscapable of both chemical reaction with the surface and also capable ofefficient UV-initiated crosslinking with the nucleic acid probe. Themechanics of this process is spontaneous 3D self assembly and isillustrated in FIG. 1A-FIG. 1D. As seen in FIG. 1A, the componentsrequired to fabricate this microarray system are:

(a) an unmodified nucleic acid probe 3 such as an oligonucleotide, PCRor isothermal amplicon, plasmid or genomic DNA;

(b) a chemically activatable surface 1 with chemically activatablegroups (designated “X”) compatible for reacting with a primary aminesuch as. epoxysilane, isocyanate, succinimide, carbodiimide, aldehyde.

(c) bifunctional polymer linkers 2 such as a natural or modifiedOligodT, amino polysaccharide, amino polypeptide suitable for couplingto chemically activatable groups on the support surface, each attachedwith a fluorescent label 4; and

(d) a solvent comprising water and a high boiling point, water-miscibleliquid such as glycerol, DMSO or propanediol (water to solvent ratiobetween 10:1 and 100:1).

Table 1 shows examples of chemically activatable groups and matchedreactive groups on the bifunctional polymer linker for mere illustrationpurposes only and does not in any way preclude use of other combinationsof matched reactive pairs.

TABLE 1 Covalent Attachment of Bifunctional Polymeric Linker to anActivated Surfaces Activated Surface Matched Reactive Group SpecificImplementation as Bifunctional Moiety on Bifunctional Linker polymericlinker Epoxysilane Primary Amine (1) Amine-modified OligodT (20-60bases) (2) Chitosan (20-60 subunits) (3) Lysine containing polypeptide(20-60aa) EDC Activated Primary Amine (4) Amine-modified OligodT (20-60bases) Carboxylic Acid (5) Chitosan (20-60 subunits) (6) Lysinecontaining polypeptide (20-60aa) N-hydroxysuccinimide Primary Amine (7)Amine-modified OligodT (20-60 bases) (NHS) (8) Chitosan (20-60 subunits)(9) Lysine containing polypeptide (20-60aa)

When used in the present invention, the chemically activatable surface,bifunctional polymer linkers and unmodified nucleic acid probes areincluded as a solution to be applied to a chemically activated surface 4by ordinary methods of fabrication used to generate DNA Hybridizationtests such as contact printing, piezo electric printing, ink jetprinting, or pipetting.

Microarray fabrication begins with application of a mixture of thechemically activatable surface, bifunctional polymer linkers andunmodified nucleic acid probes to the surface. The first step isreaction and covalent attachment of the bifunctional linker to theactivated surface (FIG. 1B). In general, the chemical concentration ofthe bi-functional linker is set to be such that less than 100% of thereactive sites on the surface form a covalent linkage to thebi-functional linker. At such low density, the average distance betweenbi-functional linker molecules defines a spacing denoted lattice width(“LW” in FIG. 1B).

In the second step, the water in the solvent is evaporated toconcentrate the DNA and bifunctional linker via evaporation of waterfrom the solvent (FIG. 1C). Generally, use of pure water as the solventduring matrix fabrication is disadvantageous because water is quicklyremoved by evaporation due to a high surface area/volume ratio. Toovercome this, in the present invention, a mixture of water with a highboiling point water-miscible solvent such as glycerin, DMSO orpropanediol was used as solvent. In this case, upon evaporation, thewater component will evaporate but not the high boiling point solvent.As a result, molecular reactants—DNA and bifunctional linker areprogressively concentrated as the water is lost to evaporation. In thepresent invention, the ratio or water to high boiling point solvent iskept between 10:1 and 100:1. Thus, in the two extreme cases, uponequilibrium, volume of the fluid phase will reduce due to waterevaporation to between 1/100th and 1/10^(th) the original volume, thusgiving rise to a 100-fold to 10-fold increase in reactant concentration.Such controlled evaporation is crucial to the present invention since itcontrols the vertical spacing (Vertical Separation, “VG” in FIG. 1C)between nucleic acid probes, which is inversely related to the extent ofevaporative concentration.

In the third step, the terminal Thymidine bases in the nucleic acidprobes are UV crosslinked to the bifunctional linker within theevaporated surface (FIG. 1D). This process is mediated by the well-knownphotochemical reactivity of the Thymidine base that leads to theformation of covalent linkages to other thymidine bases in DNA orphotochemical reaction with proteins and carbohydrates. If thebifunctional crosslinker is OligodT, then the crosslinking reaction willbe bi-directional, that is, the photochemistry can be initiated ineither the nucleic acid probe or the bifunctional OligodT linker. On theother hand, if the bifunctional linker is an amino polysaccharide suchas chitosan or a polyamino acid, with a lysine or histidine in it, thenthe photochemistry will initiate in the nucleic acid probe, with thebifunctional linker being the target of the photochemistry.

Microarray System with Unmodified Solid Support for Non-CovalentAttachment

In this microarray system, attachment of the nucleic acid probes doesnot occur to the underlying surface directly, but is instead mediatedthrough a relatively long, bi-functional polymeric linker that bindsnon-covalently with the solid support, but covalently with the nucleicacid probes via UV-initiated crosslinking. The mechanics of this processis spontaneous 3D self assembly and is illustrated in FIGS. 2A-2D. Asseen in FIG. 2A, the components required to fabricate this microarraysystem are:

-   -   (1) an unmodified nucleic acid probe 3 such as an        oligonucleotide, PCR or isothermal amplicon, plasmid or genomic        DNA;    -   (2) an unmodified solid support 1    -   (3) bifunctional polymer linkers 2 such as OligodT or a amino        polysaccharide, amino polypeptide, that inherently have or are        modified to have functional groups (designated “R”) compatible        for adsorptive binding to the solid support, each having a        fluorescent label 4; and    -   (4) a solvent comprising water and a high boiling point,        water-miscible liquid such as glycerol, DMSO or propanediol        (water to solvent ratio between 10:1 and 100:1);

TABLE 2 Non-Covalent Attachment of Bi-Functional Polymeric Linker to anInert Surface Representative Matched Adsorptive Group SpecificBifunctional support surface on Bifunctional Linker (R_(n)) polymericlinker glass Single Stranded Nucleic OligodT (30-60 bases) Acid > 10bases glass Amine-Polysaccharide Chitosan (30-60 subunits) glassExtended Planar Hydrophobic OligodT (30-60 bases)- Groups e.g.Digoxigenin 5′-Digoxigenin polycarbonate Single Stranded NucleicOligo-dT (30-60 bases) Acid > 10 bases polycarbonateAmine-Polysaccharide Chitosan (30-60 subunits) polycarbonate ExtendedPlanar Hydrophobic OligodT (30-60 bases)- Groups e.g. Digoxigenin5′-Digoxigenin graphene Extended Planar Hydrophobic OligodT (30-60bases)- Groups e.g. pyrene 5′pyrene graphene Extended Planar HydrophobicOligodT (30-60 bases)- Groups e.g. CY-5 dye 5′-CY-5 dye grapheneExtended Planar Hydrophobic OligodT (30-60 bases)- Groups e.g.Digoxigenin 5′-Digoxigenin gold Extended Planar Hydrophobic OligodT(30-60 bases)- Groups e.g. pyrene 5′pyrene gold Extended PlanarHydrophobic OligodT (30-60 bases)- Groups e.g. CY-5 dye 5′ CY-5 dye goldExtended Planar Hydrophobic OligodT (30-60 bases)- Groups e.g.Digoxigenin 5′ Digoxigenin

Table 2 shows examples of unmodified support surfaces and matchedabsorptive groups on the bifunctional polymer linker for mereillustration purposes only and does not in any way precludes the use ofother combinations of these.

When used in the present invention, components 1-3 are included as asolution to be applied to the solid support surface by ordinary methodsof fabrication used to generate DNA Hybridization tests such as contactprinting, piezo electric printing, ink jet printing, or pipetting.

Microarray fabrication begins with application of a mixture of thecomponents (1)-(3) to the surface. The first step is adsorption of thebifunctional linker to the support surface (FIG. 2B). The concentrationof the bi-functional linker is set so the average distance betweenbi-functional linker molecules defines a spacing denoted as latticewidth (“LW” in FIG. 2B).

In the second step, the water in the solvent is evaporated toconcentrate the DNA and bifunctional linker via evaporation of waterfrom the solvent (FIG. 2C). Generally, use of pure water as the solventduring matrix fabrication is disadvantageous because water is quicklyremoved by evaporation due to a high surface area/volume ratio. Toovercome this, in the present invention, a mixture of water with a highboiling point water-miscible solvent such as glycerin, DMSO orpropanediol was used as solvent. In this case, upon evaporation, thewater component will evaporate but not the high boiling point solvent.As a result, molecular reactants—DNA and bifunctional linker areprogressively concentrated as the water is lost to evaporation. In thepresent invention, the ratio or water to high boiling point solvent iskept between 10:1 and 100:1. Thus, in the two extreme cases, uponequilibrium, volume of the fluid phase will reduce due to waterevaporation to between 1/100th and 1/10^(th) the original volume, thusgiving rise to a 100-fold to 10-fold increase in reactant concentration.

In the third step, the terminal Thymidine bases in the nucleic acidprobes are UV crosslinked to the bifunctional linker within theevaporated surface (FIG. 2D). This process is mediated by the well-knownphotochemical reactivity of the Thymidine base that leads to theformation of covalent linkages to other thymidine bases in DNA orphotochemical reaction with proteins and carbohydrates. If thebifunctional crosslinker is OligodT, then the crosslinking reaction willbe bi-directional, that is, the photochemistry can be initiated ineither the nucleic acid probe or the bifunctional OligodT linker. On theother hand, if the bifunctional linker is an amino polysaccharide suchas chitosan or a polyamino acid, with a lysine or histidine in it, thenthe photochemistry will initiate in the nucleic acid probe, with thebifunctional linker being the target of the photochemistry.

Although such non-covalent adsorption described in the first step isgenerally weak and reversible, when occurring in isolation, in thepresent invention it is taught that if many such weak adsorptive eventsbetween the bifunctional polymeric linker and the underlying surfaceoccur in close proximity, and if the closely packed polymeric linkersare subsequently linked to each other via Thymidine-mediatedphotochemical crosslinking, the newly created extended, multi-molecular(crosslinked) complex will be additionally stabilized on the surface,thus creating a stable complex with the surface in the absence of directcovalent bonding to that surface.

The present invention works efficiently for the linkage of syntheticoligonucleotides as nucleic acid probes to form a microarray-basedhybridization device for the analysis of microbial DNA targets. However,it is clear that the same invention may be used to link PCR amplicons,synthetic oligonucleotides, isothermal amplification products, plasmidDNA or genomic DNA fragment as nucleic acid probes. It is also clearthat the same technology could be used to manufacture hybridizationdevices that are not microarrays.

DNA nucleic acid probes were formulated as described in Table 3, to bedeployed as described above and illustrated in FIG. 1 or FIG. 2. A setof 48 such probes (Table 4) were designed to be specific for varioussequence determinants of microbial DNA and each was fabricated so as topresent a string of 5-7 T bases at each end, to facilitate theirUV-crosslinking to form a covalently linked microarray element, asdescribed above and illustrated in FIG. 1. Each of the 48 differentprobes was printed in triplicate to form a 144 element (12×12)microarray having sequences shown in Table 3.

TABLE 3 Representative Conditions of use of the Present Invention 5′labelled Unique sequence OligodT Oligonucleotide Fluorescent Nucleicacid probe 30-38 bases Long marker 30 bases Type 7 T's at each endLong(marker) Nucleic acid probe 50 mM 0.15 mM Concentration BifunctionalLinker OligodT 30 bases long Primary amine at 3′ terminus BifunctionalLinker 1 mM Concentration High Boiling point Water:Propanediol, Solvent100:1 Surface Epoxysilane on borosilicate glass UV Crosslinking Dose 300millijoule (mjoule)

TABLE 4 Nucleic acid probes Linked to theMicroarray Surface via the Present Invention SEQ ID NO: 132Negative control TTTTTTCTACTACCTATGCTGATTCACTCTTTTT SEQ ID NO: 129Imager Calibration TTTTCTATGTATCGATGTTGAGAAATTTTTTT (High)SEQ ID NO: 130 Imager Calibration TTTTCTAGATACTTGTGTAAGTGAATTTTTTT (Low)SEQ ID NO: 131 Imager Calibration TTTTCTAAGTCATGTTGTTGAAGAATTTTTTT(Medium) SEQ ID NO: 126 Cannabis ITS1 DNATTTTTTAATCTGCGCCAAGGAACAATATTTTTTT Control 1 SEQ ID NO: 127Cannabis ITS1 DNA TTTTTGCAATCTGCGCCAAGGAACAATATTTTTT Control 2SEQ ID NO: 128 Cannabis ITS1 DNA TTTATTTCTTGCGCCAAGGAACAATATTTTATTTControl 3 SEQ ID NO: 86 Total Yeast and MoldTTTTTTTTGAATCATCGARTCTTTGAACGCATTTT (High sensitivity) TTT SEQ ID NO: 87Total Yeast and Mold TTTTTTTTGAATCATCGARTCTCCTTTTTTT (Low sensitivity)SEQ ID NO: 88 Total Yeast and Mold TTTTTTTTGAATCATCGARTCTTTGAACGTTTTTTT(Medium sensitivity) SEQ ID NO: 132 Negative controlTTTTTTCTACTACCTATGCTGATTCACTCTTTTT SEQ ID NO: 92 Aspergillus fumigatus 1TTTCTTTTCGACACCCAACTTTATTTCCTTATTT SEQ ID NO: 90 Aspergillus flavus 1TTTTTTCGCAAATCAATCTTTTTCCAGTCTTTTT SEQ ID NO: 95 Aspergillus niger 1TTTTTTCGACGTTTTCCAACCATTTCTTTT SEQ ID NO: 100 Botrytis spp.TTTTTTTCATCTCTCGTTACAGGTTCTCGGTTCTT TTTTT SEQ ID NO: 108 Fusarium spp.TTTTTTTTAACACCTCGCRACTGGAGATTTTTTT SEQ ID NO: 89 Alternaria sppTTTTTTCAAAGGTCTAGCATCCATTAAGTTTTTT SEQ ID NO: 123 Rhodoturula spp.TTTTTTCTCGTTCGTAATGCATTAGCACTTTTTT SEQ ID NO: 117 Penicillium paxilliTTTTTTCCCCTCAATCTTTAACCAGGCCTTTTTT SEQ ID NO: 116 Penicillium oxalicumTTTTTTACACCATCAATCTTAACCAGGCCTTTTT SEQ ID NO: 118 Penicillium spp.TTTTTTCAACCCAAATTTTTATCCAGGCCTTTTT SEQ ID NO: 102 Candida spp. Group 1TTTTTTTGTTTGGTGTTGAGCRATACGTATTTTT SEQ ID NO: 103 Candida spp. Group 2TTTTACTGTTTGGTAATGAGTGATACTCTCATTTT SEQ ID NO: 124 Stachybotrys sppTTTCTTCTGCATCGGAGCTCAGCGCGTTTTATTT SEQ ID NO: 125 Trichoderma spp.TTTTTCCTCCTGCGCAGTAGTTTGCACATCTTTT SEQ ID NO: 105 Cladosporium spp.TTTTTTTTGTGGAAACTATTCGCTAAAGTTTTTT SEQ ID NO: 121 Podosphaera spp.TTTTTTTTAGTCAYGTATCTCGCGACAGTTTTTT SEQ ID NO: 132 Negative controlTTTTTTCTACTACCTATGCTGATTCACTCTTTTT SEQ ID NO: 37 Total Aerobic bacteriaTTTTTTTTTCCTACGGGAGGCAGTTTTTTT (High) SEQ ID NO: 38Total Aerobic bacteria TTTTTTTTCCCTACGGGAGGCATTTTTTTT (Medium)SEQ ID NO: 39 Total Aerobic bacteria TTTATTTTCCCTACGGGAGGCTTTTATTTT(Low) SEQ ID NO: 47 Bile-tolerant Gram-TTTTTCTATGCAGTCATGCTGTGTGTRTGTCTTTT negative (High) T SEQ ID NO: 48Bile-tolerant Gram- TTTTTCTATGCAGCCATGCTGTGTGTRTTTTTTT negative (Medium)SEQ ID NO: 49 Bile-tolerant Gram- TTTTTCTATGCAGTCATGCTGCGTGTRTTTTTTTnegative (Low) SEQ ID NO: 53 Coliform/ TTTTTTCTATTGACGTTACCCGCTTTTTTTEnterobacteriaceae SEQ ID NO: 81 stx1 geneTTTTTTCTTTCCAGGTACAACAGCTTTTTT SEQ ID NO: 82 stx2 geneTTTTTTGCACTGTCTGAAACTGCCTTTTTT SEQ ID NO: 59 etuf geneTTTTTTCCATCAAAGTTGGTGAAGAATCTTTTTT SEQ ID NO: 132 Negative controlTTTTTTCTACTACCTATGCTGATTCACTCTTTTT SEQ ID NO: 65 Listeria spp.TTTTCTAAGTACTGTTGTTAGAGAATTTTT SEQ ID NO: 56 Aeromonas spp.TTATTTTCTGTGACGTTACTCGCTTTTATT SEQ ID NO: 78 Staphylococcus aureusTTTATTTTCATATGTGTAAGTAACTGTTTTATTT 1 SEQ ID NO: 49 Campylobacter spp.TTTTTTATGACACTTTTCGGAGCTCTTTTT SEQ ID NO: 72 Pseudomonas spp. 3TTTATTTTAAGCACTTTAAGTTGGGATTTTATTT SEQ ID NO: 53 Clostridium spp.TTTTCTGGAMGATAATGACGGTACAGTTTT SEQ ID NO: 42 Escherichia coli/TTTTCTAATACCTTTGCTCATTGACTCTTT Shigella 1 SEQ ID NO: 74Salmonella enterica/ TTTTTTTGTTGTGGTTAATAACCGATTTTT Enterobacter 1SEQ ID NO: 61 invA gene TTTTTTTATTGATGCCGATTTGAAGGCCTTTTTT

The set of 48 different probes of Table 4 were formulated as describedin Table 3, then printed onto epoxysilane coated borosilicate glass,using an Gentics Q-Array mini contact printer with Arrayit SMP pins,which deposit about 1 nL of formulation per spot. As described in FIG.1, the arrays thus printed were then allowed to react with theepoxisilane surface at room temperature, and then evaporate to removefree water, also at room temperature. Upon completion of the evaporationstep (typically overnight) the air-dried microarrays were then UVtreated in a STATOLINKER® UV irradiation system: 300 mjoules ofirradiation at 254 nm to initiate thymidine-mediated crosslinking. Themicroarrays are then ready for use, with no additional need for washingor capping.

Example 2

Using the 3-Dimensional Lattice Microarray System for DNA Analysis

Sample Processing

Harvesting Pathogens from plant surface comprises the following steps:

-   -   1. Wash the plant sample or tape pull in 1× phosphate buffered        saline (PBS);    -   2. Remove plant material/tape;    -   3. Centrifuge to pellet cells and discard supernatant;    -   4. Resuspend in PathogenDx® Sample Prep Buffer pre-mixed with        Sample        Digestion Buffer;    -   5. Heat at 55° C. for 45 minutes;    -   6. Vortex to dissipate the pellet;    -   7. Heat at 95° C. for 15 minutes; and    -   8. Vortex and centrifuge briefly before use in PCR.        Amplification by PCR

The sample used for amplification and hybridization analysis was aCannabis flower wash from a licensed Cannabis lab. The washed flowermaterial was then pelleted by centrifugation. The pellet was thendigested with proteinase K, then spiked with a known amount ofSalmonella DNA before PCR amplification.

TABLE 5 PCR Primers and PCR conditions used in amplificationPCR primers (P1) for PCR Reaction #1 Cannabis ITS1 1° FP*-(SEQ ID NO: 17) TTTGCAACAGCAGAACGACCCGTGA Cannabis ITS1 1° RP*-(SEQ ID NO: 18) TTTCGATAAACACGCATCTCGATTG Enterobacteriaceae 16S 1° FP-(SEQ ID NO: 11) TTACCTTCGGGCCTCTTGCCATCRGATGTG Enterobacteriaceae 16S 1°RP- (SEQ ID NO: 12) TTGGAATTCTACCCCCCTCTACRAGACTCAAGCPCR primers (P2) for PCR Reaction #2 Cannabis ITS1 2° FP- (SEQ ID NO: 5)TTTCGTGAACACGTTTTAAACAGCTTG Cannabis ITS1 2° RP- (SEQ ID NO: 36)(CY3)TTTCCACCGCACGAGCCACGCGAT Enterobacteriaceae 16S 2° FP-(SEQ ID NO: 29) TTATATTGCACAATGGGCGCAAGCCTGATG Enterobacteriaceae 16S 2°RP- (SEQ ID NO: 30) (CY3)TTTTGTATTACCGCGGCTGCTGGCA Primary PCRSecondary PCR PCR Reagent Concentration Concentration PCR Buffer 1X 1XMgCl₂  2.5 mM    2.5 mM BSA 0.16 mg/mL   0.16 mg/mL dNTP's  200 mM   200 mM Primer mix  200 nM each     50 nM-FP/    200 nM RP Taq 1.5 Units    1.5 Units Polymerase Program for PCR Reaction #1 95° C.,98° C., 30s 61° C., 30s 72° C., 60s 72° C., 4 min 25X 7 minProgram for PCR Reaction #2 95° C., 98° C., 20s 61° C., 20s 72° C., 30s72° C., 4 min 25X 7 min *FP, Forward Primer; *RP, Reverse Primer

The Salmonella DNA spiked sample was then amplified with PCR primers(P1-Table 5) specific for the 16s region of Enterobacteriaceae in atandem PCR reaction to first isolate the targeted region (PCR Reaction#1) and also PCR primers (P1-Table 5) which amplify a segment ofCannabis DNA (ITS) used as a positive control.

The product of PCR Reaction #1 (1 μL) was then subjected to a second PCRreaction (PCR Reaction #2) which additionally amplified and labelled thetwo targeted regions (16s, ITS) with green CY3 fluorophore labeledprimers (P2-Table 5). The product of the PCR Reaction #2 (504) was thendiluted 1-1 with hybridization buffer (4×SSC+5×Denhardt's solution) andthen applied directly to the microarray for hybridization.

Hybridization

Because the prior art method of microarray manufacture allows DNA to beanalyzed via hybridization without the need for pre-treatment of themicroarray surface, the use of the microarray is simple, and involves 6manual or automated pipetting steps.

1) Pipette the amplified DNA+binding buffer onto the microarray

2) Incubate for 30 minutes to allow DNA binding to the microarray(typically at room temperature, RT)

3) Remove the DNA+binding buffer by pipetting

4) Pipette 50 uL of wash buffer onto the microarray(0.4×SSC+0.5×Denhardt's) and incubate 5 min at RT.

5) Remove the wash buffer by pipetting

6) Repeat steps 4 and 5

7) Perform image analysis at 532 nm and 635 nm to detect the probe spotlocation (532 nm) and PCR product hybridization (635 nm).

Image Analysis

Image Analysis was performed at two wavelengths (532 nm and 635 nm) on araster-based confocal scanner: GenePix 4000B Microarray Scanner, withthe following imaging conditions: 33% Laser power, 400 PMT setting at532 nm/33% Laser Power, 700 PMT setting at 635 nm. FIG. 3 shows anexample of the structure and hybridization performance of themicroarray.

FIG. 3A reveals imaging of the representative microarray, describedabove, after hybridization and washing, as visualized at 635 nm. The 635nm image is derived from signals from the (red) CY5 fluor attached tothe 5′ terminus of the bifunctional polymer linker OligodT which hadbeen introduced during microarray fabrication as a positional marker ineach microarray spot (see FIG. 1 and Table 3). The data in FIG. 3Aconfirm that the CY5-labelled OligodT has been permanently linked to themicroarray surface, via the combined activity of the bi-functionallinker and subsequent UV-crosslinking, as described in FIG. 1.

FIG. 3B reveals imaging of the representative microarray described aboveafter hybridization and washing as visualized at 532 nm. The 532 nmimage is derived from signals from the (green) CY3 fluor attached to the5′ terminus of PCR amplified DNA obtained during PCR Reaction #2. It isclear from FIG. 3B that only a small subset of the 48 discrete probesbind to the CY3-labelled PCR product, thus confirming that the presentmethod of linking nucleic acid probes to form a microarray (FIG. 1)yields a microarray product capable of sequence specific binding to a(cognate) solution state target. The data in FIG. 3B reveal theunderlying 3-fold repeat of the data (i.e., the array is the same set of48 probes printed three times as 3 distinct sub-arrays to form the final48×3=144 element microarray. The observation that the same set of 48probes can be printed 3-times, as three repeated sub-domains show thatthe present invention generates microarray product that is reproducible.

FIG. 3C reveals imaging of the representative microarray, describedabove, after hybridization and washing, as visualized with both the 532nm and 635 nm images superimposed. The superimposed images display theutility of parallel attachment of a CY5-labelled OligodT positionalmarker relative to the sequence specific binding of the CY3-labelled PCRproduct.

Example 3

First PCR Amplification Step

FIG. 4A shows an exemplar of the first PCR step. As is standard, suchPCR reactions are initiated by the administration of PCR Primers.Primers define the start and stopping point of the PCR based DNAamplification reaction. In this embodiment, a pair of PCR reactions isutilized to support the needed DNA amplification. In general, such PCRamplification is performed in series: a first pair of PCRs, with thesuffix “P1” in FIG. 4A are used to amplify about 1 μL of any unpurifiedDNA sample, such as a raw Cannabis leaf wash for example. About 1 μL ofthe product of that first PCR reaction is used as the substrate for asecond PCR reaction that is used to affix a fluorescent dye label to theDNA, so that the label may be used to detect the PCR product when itbinds by hybridization to the microarray. The primer sequences for thefirst and second PCRs are shown in Table 6. The role of this two-stepreaction is to avert the need to purify the pathogen DNA to be analyzed.The first PCR reaction, with primers “P1” is optimized to accommodatethe raw starting material, while the second PCR primer pairs “P2” areoptimized to obtain maximal DNA yield, plus dye labeling from theproduct of the first reaction. Taken in the aggregate, the sum of thetwo reactions obviates the need to either purify or characterize thepathogen DNA of interest.

FIG. 4A reveals at low resolution the 16s rDNA region which is amplifiedin an embodiment, to isolate and amplify a region which may besubsequently interrogated by hybridization. The DNA sequence of this 16srDNA region is known to vary greatly among different bacterial species.Consequently, having amplified this region by two step PCR, thatsequence variation may be interrogated by the subsequent microarrayhybridization step.

TABLE 6 First and Second PCR Primers SEQ ID NO. Primer targetPrimer sequence First PCR Primers (P1) for the first amplification stepSEQ ID NO: 1 16s rDNA HV3 TTTCACAYTGGRACTGAGACACG Locus (Bacteria)SEQ ID NO: 2 16s rDNA HV3 TTTGACTACCAGGGTATCTAATCCTGT Locus (Bacteria)SEQ ID NO: 3 Stx1 Locus TTTATAATCTACGGCTTATTGTTGAACG(Pathogenic E. coli) SEQ ID NO: 4 Stx1 LocusTTTGGTATAGCTACTGTCACCAGACAATG (Pathogenic E. coli) SEQ ID NO: 5Stx2 Locus TTTGATGCATCCAGAGCAGTTCTGCG (Pathogenic E. coli) SEQ ID NO: 6Stx2 Locus TTTGTGAGGTCCACGTCTCCCGGCGTC (Pathogenic E. coli) SEQ ID NO: 7InvA Locus TTTATTATCGCCACGTTCGGGCAATTCG (Salmonella) SEQ ID NO: 8InvA Locus TTTCTTCATCGCACCGTCAAAGGAACCG (Salmonella) SEQ ID NO: 9tuf Locus  TTTCAGAGTGGGAAGCGAAAATCCTG (All E. coli) SEQ ID NO: 10tuf Locus  TTTACGCCAGTACAGGTAGACTTCTG (All E. coli) SEQ ID NO: 1116s rDNA TTACCTTCGGGCCTCTTGCCATCRGATGTG Enterobacteriaceae HV3 LocusSEQ ID NO: 12 16s rDNA TTGGAATTCTACCCCCCTCTACRAGACTCAAGCEnterobacteriaceae HV3 Locus SEQ ID NO: 13 ITS2 LocusTTTACTTTYAACAAYGGATCTCTTGG (All Yeast, Mold/Fungus) SEQ ID NO: 14ITS2 Locus TTTCTTTTCCTCCGCTTATTGATATG (All Yeast, Mold/Fungus)SEQ ID NO: 15 ITS2 Locus TTTAAAGGCAGCGGCGGCACCGCGTCCG (Aspergillusspecies) SEQ ID NO: 16 ITS2 Locus TTTTCTTTTCCTCCGCTTATTGATATG(Aspergillus species) SEQ ID NO: 17 ITS1 Locus TTTGCAACAGCAGAACGACCCGTGA(Cannabis/Plant) SEQ ID NO: 18 ITS1 Locus TTTCGATAAACACGCATCTCGATTG(Cannabis/Plant)Second PCR Primers (P2) for the second labeling amplification stepSEQ ID NO: 19 16s rDNA HV3 TTTACTGAGACACGGYCCARACTC Locus (All Bacteria)SEQ ID NO: 20 16s rDNA HV3 TTTGTATTACCGCGGCTGCTGGCA Locus (All Bacteria)SEQ ID NO: 21 Stx1 Locus TTTATGTGACAGGATTTGTTAACAGGAC(Pathogenic E. coli) SEQ ID NO: 22 Stx1 LocusTTTCTGTCACCAGACAATGTAACCGCTG (Pathogenic E. coli) SEQ ID NO: 23Stx2 Locus TTTTGTCACTGTCACAGCAGAAG (Pathogenic E. coli) SEQ ID NO: 24Stx2 Locus TTTGCGTCATCGTATACACAGGAGC (Pathogenic E. coli) SEQ ID NO: 25InvA Locus (All TTTTATCGTTATTACCAAAGGTTCAG Salmonella) SEQ ID NO: 26InvA Locus (All TTTCCTTTCCAGTACGCTTCGCCGTTCG Salmonella) SEQ ID NO: 27tuf Locus (All E. TTTGTTGTTACCGGTCGTGTAGAAC coli) SEQ ID NO: 28tuf Locus (All E. TTTCTTCTGAGTCTCTTTGATACCAACG coli) SEQ ID NO: 2916s rDNA TTATATTGCACAATGGGCGCAAGCCTGATG Enterobacteriaceae HV3 LocusSEQ ID NO: 30 16s rDNA TTTTGTATTACCGCGGCTGCTGGCA EnterobacteriaceaeHV3 Locus SEQ ID NO: 31 ITS2 Locus (All TTTGCATCGATGAAGARCGYAGC Yeast,Mold/Fungus) SEQ ID NO: 32 ITS2 Locus (All TTTCCTCCGCTTATTGATATGC Yeast,Mold/Fungus) SEQ ID NO: 33 ITS2 Locus TTTCCTCGAGCGTATGGGGCTTTGTC(Aspergillus species) SEQ ID NO: 34 ITS2 Locus TITTTCCTCCGCTTATIGATATGC(Aspergillus species) SEQ ID NO: 35 ITS1 LocusTTTCGTGAACACGTTTTAAACAGCTTG (Cannabis/Plant) SEQ ID NO: 36 ITS1 LocusTTTCCACCGCACGAGCCACGCGAT (Cannabis/Plant)

FIG. 4B displays the stx1 gene locus which is present in the mostimportant pathogenic strains of E coli and which encodes Shigatoxin 1.Employing the same two-step PCR approach, a set of two PCR primer pairswere designed which, in tandem, can be used to amplify and labelunprocessed bacterial samples to present the stx1 locus for analysis bymicroarray-based DNA hybridization.

FIG. 5A displays the stx2 gene locus which is also present in the mostimportant pathogenic strains of E coli and which encodes Shigatoxin 2.Employing the same two-step PCR approach, a set of two PCR primer pairswere designed which, in tandem, can be used to amplify and labelunprocessed bacterial samples so as to present the stx2 locus foranalysis by microarray-based DNA hybridization.

FIG. 5B displays the invA gene locus which is present in all strains ofSalmonella and which encodes the InvAsion A gene product. Employing thesame two-step PCR approach, a set of two PCR primer pairs were designedwhich, in tandem, can be used to amplify and label unprocessed bacterialsamples so as to present the invA locus for analysis by microarray-basedDNA hybridization.

FIG. 6 displays the tuf gene locus which is present in all strains of Ecoli and which encodes the ribosomal elongation factor Tu. Employing thesame two-step PCR approach, a set of two PCR primer pairs were designedwhich, in tandem, can be used to amplify and label unprocessed bacterialsamples so as to present the tuf locus for analysis by microarray-basedDNA hybridization.

FIG. 7 displays the ITS2 locus which is present in all eukaryotes,including all strains of yeast and mold and which encodes the intergenicregion between ribosomal genes 5.8S and 28S. ITS2 is highly variable insequence and that sequence variation can be used to resolve straindifferences in yeast, and mold. Employing the same two-step PCRapproach, a set of two PCR primer pairs were designed which, in tandem,can be used to amplify and label unprocessed yeast and mold samples soas to present the ITS2 locus for analysis by microarray-based DNAhybridization.

FIG. 8 displays the ITS1 gene locus which is present in all eukaryotes,including all plants and animals, which encodes the intergenic regionbetween ribosomal genes 18S and 5.8S. ITS1 is highly variable insequence among higher plants and that sequence variation can be used toidentify plant species. Employing the same two-step PCR approach, a setof two PCR primer pairs were designed which, in tandem, can be used toamplify and label unprocessed Cannabis samples so as to present the ITS1locus for analysis by microarray-based DNA hybridization. Theidentification and quantitation of the Cannabis sequence variant of ITS1is used as an internal normalization standard in the analysis ofpathogens recovered from the same Cannabis samples.

Table 7 displays representative oligonucleotide sequences which are usedas microarray probes in an embodiment for DNA microarray-based analysisof bacterial 16s locus as described in FIG. 4. The sequence of thoseprobes has been varied to accommodate the cognate sequence variationwhich occurs as a function of species difference among bacteria. In allcases, the probe sequences are terminated with a string of T's at eachend, to enhance the efficiency of probe attachment to the microarraysurface, at time of microarray manufacture. Table 8 shows sequences ofthe Calibration and Negative controls used in the microarray.

Table 9 displays representative oligonucleotide sequences which are usedas microarray probes in an embodiment for DNA microarray-based analysisof eukaryotic pathogens (fungi, yeast and mold) based on their ITS2locus as described in FIG. 7. Sequences shown in Table 8 are used ascontrols. The sequence of those probes has been varied to accommodatethe cognate sequence variation which occurs as a function of speciesdifference among fungi, yeast and mold. In all cases, the probesequences are terminated with a string of T's at each end, to enhancethe efficiency of probe attachment to the microarray surface, at time ofmicroarray manufacture.

Table 10 displays representative oligonucleotide sequences which areused as microarray probes in an embodiment for DNA microarray-basedanalysis of Cannabis at the ITS1 locus (Cannabis spp.).

TABLE 7 Oligonucleotide probe sequence for the 16S Locus SEQ ID NO: 37Total Aerobic bacteria TTTTTTTTTCCTACGGGAGGCAGTTTTTTT (High)SEQ ID NO: 38 Total Aerobic bacteria TTTTTTTTCCCTACGGGAGGCATTTTTTTT(Medium) SEQ ID NO: 39 Total Aerobic bacteriaTTTATTTTCCCTACGGGAGGCTTTTATTTT (Low) SEQ ID NO: 40 EnterobacteriaceaeTTTATTCTATTGACGTTACCCATTTATTTT (Low sensitivity) SEQ ID NO: 41Enterobacteriaceae TTTTTTCTATTGACGTTACCCGTTTTTTTT (Medium sensitivity)SEQ ID NO: 42 Escherichia coli/ TTTTCTAATACCTTTGCTCATTGACTCTTTShigella 1 SEQ ID NO: 43 Escherichia coli/TTTTTTAAGGGAGTAAAGTTAATATTTTTT Shigella 2 SEQ ID NO: 44Escherichia coli/ TTTTCTCCTTTGCTCATTGACGTTATTTTT Shigella 3SEQ ID NO: 45 Bacillus spp. Group1 TTTTTCAGTTGAATAAGCTGGCACTCTTTTSEQ ID NO: 46 Bacillus spp. Group2 TTTTTTCAAGTACCGTTCGAATAGTTTTTTSEQ ID NO: 47 Bile-tolerant Gram- TTTTTCTATGCAGTCATGCTGTGTGTRTGTCTTnegative (High) TTT SEQ ID NO: 48 Bile-tolerant Gram-TTTTTCTATGCAGCCATGCTGTGTGTRTTTTTTT negative (Medium) SEQ ID NO: 49Bile-tolerant Gram- TTTTTCTATGCAGTCATGCTGCGTGTRTTTTTTT negative (Low)SEQ ID NO: 50 Campylobacter spp. TTTTTTATGACACTTTTCGGAGCTCTTTTTSEQ ID NO: 51 Chromobacterium spp. TTTTATTTTCCCGCTGGTTAATACCCTTTATTTTSEQ ID NO: 52 Citrobacter spp. TTTTTTCCTTAGCCATTGACGTTATTTTTT Group1SEQ ID NO: 53 Clostridium spp. TTTTCTGGAMGATAATGACGGTACAGTTTTSEQ ID NO: 54 Coliform/Entero- TTTTTTCTATTGACGTTACCCGCTTTTTTTbacteriaceae SEQ ID NO: 55 Aeromonas TTTTTGCCTAATACGTRTCAACTGCTTTTTsalmonicida/hydrophilia SEQ ID NO: 56 Aeromonas spp.TTATTTTCTGTGACGTTACTCGCTTTTATT SEQ ID NO: 57 Alkanindiges spp.TTTTTAGGCTACTGRTACTAATATCTTTTT SEQ ID NO: 58 Bacillus pumilusTTTATTTAAGTGCRAGAGTAACTGCTATTTTATT SEQ ID NO: 59 etuf geneTTTTTTCCATCAAAGTTGGTGAAGAATCTTTTTT SEQ ID NO: 60 Hafnia spp.TTTTTTCTAACCGCAGTGATTGATCTTTTT SEQ ID NO: 61 invA geneTTTTTTTATTGATGCCGATTTGAAGGCCTTTTTT SEQ ID NO: 62 Klebsiella oxytocaTTTTTTCTAACCTTATTCATTGATCTTTTT SEQ ID NO: 63 Klebsiella pneumoniaeTTTTTTCTAACCTTGGCGATTGATCTTTTT SEQ ID NO: 64 Legionella spp.TTTATTCTGATAGGTTAAGAGCTGATCTTTATTT SEQ ID NO: 65 Listeria spp.TTTTCTAAGTACTGTTGTTAGAGAATTTTT SEQ ID NO: 66 Panteoa agglomeransTTTTTTAACCCTGTCGATTGACGCCTTTTT SEQ ID NO: 67 Panteoa stewartiiTTTTTTAACCTCATCAATTGACGCCTTTTT SEQ ID NO: 68 PseudomonasTTTTTGCAGTAAGTTAATACCTTGTCTTTT aeruginosa SEQ ID NO: 69 PseudomonasTTTTTTTACGTATCTGTTTTGACTCTTTTT cannabina SEQ ID NO: 70Pseudomonas spp. 1 TTTTTTGTTACCRACAGAATAAGCATTTTT SEQ ID NO: 71Pseudomonas spp. 2 TTTTTTAAGCACTTTAAGTTGGGATTTTTT SEQ ID NO: 72Pseudomonas spp. 3 TTTATTTTAAGCACTTTAAGTTGGGATTTTATTT SEQ ID NO: 73Salmonella bongori TTTTTTTAATAACCTTGTTGATTGTTTTTT SEQ ID NO: 74Salmonella TTTTTTTGTTGTGGTTAATAACCGATTTTT enterica/Enterobacter 1SEQ ID NO: 75 Salmonella TTTTTTTAACCGCAGCAATTGACTCTTTTTenterica/Enterobacter 2 SEQ ID NO: 76 SalmonellaTTTTTTCTGTTAATAACCGCAGCTTTTTTT enterica/Enterobacter 3 SEQ ID NO: 77Serratia spp. TTTATTCTGTGAACTTAATACGTTCATTTTTATT SEQ ID NO: 78Staphylococcus aureus TTTATTTTCATATGTGTAAGTAACTGTTTTATTT 1 SEQ ID NO: 79Staphylococcus aureus TTTTTTCATATGTGTAAGTAACTGTTTTTT 2 SEQ ID NO: 80Streptomyces spp. TTTTATTTTAAGAAGCGAGAGTGACTTTTATTTT SEQ ID NO: 81stx1 gene TTTTTTCTTTCCAGGTACAACAGCTTTTTT SEQ ID NO: 82 stx2 geneTTTTTTGCACTGTCTGAAACTGCCTTTTTT SEQ ID NO: 83 Vibrio spp.TTTTTTGAAGGTGGTTAAGCTAATTTTTTT SEQ ID NO: 84 Xanthamonas spp.TTTTTTGTTAATACCCGATTGTTCTTTTTT SEQ ID NO: 85 Yersinia pestisTTTTTTTGAGTTTAATACGCTCAACTTTTT

TABLE 8 Calibration and Negative Controls SEQ ID NO: 129 ImagerTTTTCTATGTATCGATGT Calibration TGAGAAATTTTTTT (High) SEQ ID NO: 130Imager TTTTCTAGATACTTGTGT Calibration AAGTGAATTTTTTT (Low)SEQ ID NO: 131 Imager TTTTCTAAGTCATGTTGT Calibration TGAAGAATTTTTTT(Medium) SEQ ID NO: 132 Negative TTTTTTCTACTACCTATG controlCTGATTCACTCTTTTT

TABLE 9 Oligonucleotide probe sequence for the ITS2 Locus SEQ ID NO: 86Total Yeast TTTTTTTTGAATCATCGARTCTTTGAACGCATTTTTTT and Mold (Highsensitivity) SEQ ID NO: 87 Total Yeast TTTTTTTTGAATCATCGARTCTCCTTTTTTTand Mold (Low sensitivity) SEQ ID NO: 88 Total YeastTTTTTTTTGAATCATCGARTCTTTGAACGTTTTTTT and Mold (Medium sensitivity)SEQ ID NO: 89 Alternaria spp. TTTTTTCAAAGGTCTAGCATCCATTAAGTTTTTTSEQ ID NO: 90 Aspergillus TTTTTTCGCAAATCAATCTTTTTCCAGTCTTTTT flavus 1SEQ ID NO: 91 Aspergillus TTTTTTTCTTGCCGAACGCAAATCAATCTTTTTTTTTTflavus 2 TT SEQ ID NO: 92 Aspergillus TTTCTTTTCGACACCCAACTTTATTTCCTTATTTfumigatus 1 SEQ ID NO: 93 Aspergillus TTTTTTTGCCAGCCGACACCCATTCTTTTTfumigatus 2 SEQ ID NO: 94 Aspergillus TTTTTTGGCGTCTCCAACCTTACCCTTTTTnidulans SEQ ID NO: 95 Aspergillus TTTTTTCGACGTTTTCCAACCATTTCTTTTniger 1 SEQ ID NO: 96 Aspergillus TTTTTTTTCGACGTTTTCCAACCATTTCTTTTTTniger 2 SEQ ID NO: 97 Aspergillus TTTTTTTCGCCGACGTTTTCCAATTTTTTT niger 3SEQ ID NO: 98 Aspergillus TTTTTCGACGCATTTATTTGCAACCCTTTT terreusSEQ ID NO: 99 Blumeria TTTATTTGCCAAAAMTCCTTAATTGCTCTTTTTT SEQ ID NO: 100Botrytis spp TTTTTTTCATCTCTCGTTACAGGTTCTCGGTTCTTTTT TT SEQ ID NO: 101Candida TTTTTTTTTGAAAGACGGTAGTGGTAAGTTTTTT albicans SEQ ID NO: 102Candida spp. TTTTTTTGTTTGGTGTTGAGCRATACGTATTTTT Group 1 SEQ ID NO: 103Candida spp. TTTTACTGTTTGGTAATGAGTGATACTCTCATTTT Group 2 SEQ ID NO: 104Chaetomium TTTCTTTTGGTTCCGGCCGTTAAACCATTTTTTT spp. SEQ ID NO: 105Cladosporium TTTTTTTTGTGGAAACTATTCGCTAAAGTTTTTT spp SEQ ID NO: 106Erysiphe spp. TTTCTTTTTACGATTCTCGCGACAGAGTTTTTTT SEQ ID NO: 107 FusariumTTTTTTTCTCGTTACTGGTAATCGTCGTTTTTTT oxysporum SEQ ID NO: 108 Fusarium sppTTTTTTTTAACACCTCGCRACTGGAGATTTTTTT SEQ ID NO: 109 GolovinomycesTTTTTTCCGCTTGCCAATCAATCCATCTCTTTTT SEQ ID NO: 110 HistoplasmaTTTATTTTTGTCGAGTTCCGGTGCCCTTTTATTT capsulatum SEQ ID NO: 111 Isaria spp.TTTATTTTTCCGCGGCGACCTCTGCTCTTTATTT SEQ ID NO: 112 MonocilliumTTTCTTTTGAGCGACGACGGGCCCAATTTTCTTT spp. SEQ ID NO: 113 Mucor spp.TTTTCTCCAVVTGAGYACGCCTGTTTCTTTT SEQ ID NO: 114 MyrotheciumTTTATTTTCGGTGGCCATGCCGTTAAATTTTATT spp. SEQ ID NO: 115 OidiodendronTTTTTTTGCGTAGTACATCTCTCGCTCATTTTTT spp. SEQ ID NO: 116 PenicilliumTTTTTTACACCATCAATCTTAACCAGGCCTTTTT oxalicum SEQ ID NO: 117 PenicilliumTTTTTTCCCCTCAATCTTTAACCAGGCCTTTTTT paxilli SEQ ID NO: 118Penicillium spp TTTTTTCAACCCAAATTTTTATCCAGGCCTTTTT SEQ ID NO: 119 Phoma/TTTTTTTGCAGTACATCTCGCGCTTTGATTTTTT Epicoccum spp. SEQ ID NO: 120Podosphaera TTTTTTGACCTGCCAAAACCCACATACCATTTTT spp SEQ ID NO: 121Podosphaera TTTTTTTTAGTCAYGTATCTCGCGACAGTTTTTT spp. SEQ ID NO: 122Pythium TTTTATTTAAAGGAGACAACACCAATTTTTATTT oligandrum SEQ ID NO: 123Rhodoturula TTTTTTCTCGTTCGTAATGCATTAGCACTTTTTT spp SEQ ID NO: 124Stachybotrys TTTCTTCTGCATCGGAGCTCAGCGCGTTTTATTT spp SEQ ID NO: 125Trichoderma TTTTTCCTCCTGCGCAGTAGTTTGCACATCTTTT spp

Table 11 displays representative oligonucleotide sequences which areused as microarray probes in an embodiment for DNA microarray-basedanalysis of bacterial pathogens (stx1, stx2, invA, tuf) and for DNAanalysis of the presence host Cannabis at the ITS1 locus (Cannabisspp.). It should be noted that this same approach, with modifications tothe ITS1 sequence, could be used to analyze the presence of other planthosts in such extracts.

TABLE 10 Oligonucleotide probe sequence for the Cannabis ITS1 LocusSEQ ID NO: 126 Cannabis ITS1 TTTTTTAATCTGCGCCA DNA Control 1AGGAACAATATTTTTTT SEQ ID NO: 127 Cannabis ITS1 TTTTTGCAATCTGCGCCDNA Control 2 AAGGAACAATATTTTTT SEQ ID NO: 128 Cannabis ITS1TTTATTTCTTGCGCCAA DNA Control 3 GGAACAATATTTTATTT

TABLE 11 Representative Microarray ProbeDesign for the Present Invention: Bacterial Toxins, ITS1 (Cannabis)SEQ ID NO: 81 stx1 gene TTTTTTCTTTCCAGGTA CAACAGCTTTTTT SEQ ID NO: 82stx2 gene TTTTTTGCACTGTCTGA AACTGCCTTTTTT SEQ ID NO: 59 etuf geneTTTTTTCCATCAAAGTT GGTGAAGAATCTTTTTT SEQ ID NO: 61 invA geneTTTTTTTATTGATGCCG ATTTGAAGGCCTTTTTT SEQ ID NO: 126 Cannabis ITS1TTTTTTAATCTGCGCCA DNA Control 1 AGGAACAATATTTTTTT

FIG. 9 shows a flow diagram to describe how an embodiment is used toanalysis the bacterial pathogen or yeast and mold complement of aCannabis or related plant sample. Pathogen samples can be harvested fromCannabis plant material by tape pulling of surface bound pathogen or bysimple washing of the leaves or buds or stems, followed by a singlemultiplex “Loci Enhancement” Multiplex PCR reaction, which is thenfollowed by a single multiplex “Labelling PCR”. A different pair of twostep PCR reactions is used to analyze bacteria, than the pair of twostep PCR reactions used to analyze fungi, yeast and mold. In all cases,the DNA of the target bacteria or fungi, yeast and mold are PCRamplified without extraction or characterization of the DNA prior to twostep PCR. Subsequent to the Loci Enhancement and Labelling PCR steps,the resulting PCR product is simply diluted into binding buffer and thenapplied to the microarray test. The subsequent microarray steps requiredfor analysis (hybridization and washing) are performed at lab ambienttemperature.

FIG. 10 provide images of a representative implementation of microarraysused in an embodiment. In this implementation, all nucleic acid probesrequired for bacterial analysis, along with Cannabis DNA controls(Tables 7 and 10) are fabricated into a single 144 element (12×12)microarray, along with additional bacterial and Cannabis probes such asthose in Table 10. In this implementation, all nucleic acid probesrequired for fungi, yeast and mold analysis along with Cannabis DNAcontrols were fabricated into a single 144 element (12×12) microarray,along with additional fungal probes shown in Table 9. The arrays aremanufactured on PTFE coated glass slides as two columns of 6 identicalmicroarrays. Each of the 12 identical microarrays is capable ofperforming, depending on the nucleic acid probes employed, a completemicroarray-based analysis bacterial analysis or a completemicroarray-based analysis of fungi, yeast and mold. Nucleic acid probeswere linked to the glass support via microfluidic printing, eitherpiezoelectric or contact based or an equivalent. The individualmicroarrays are fluidically isolated from the other 11 in this case, bythe hydrophobic PTFE coating, but other methods of physical isolationcan be employed.

FIGS. 11A-11B display representative DNA microarray analysis of anembodiment. In this case, purified bacterial DNA or purified fungal DNAhas been used, to test for affinity and specificity subsequent to thetwo-step PCR reaction and microarray-based hybridization analysis. Ascan be seen, the nucleic acid probes designed to detect each of thebacterial DNA (top) or fungal DNA (bottom) have bound to the target DNAcorrectly via hybridization and thus have correctly detected thebacterium or yeast (Table 12 and 13). FIG. 12 displays representativeDNA microarray analysis of an embodiment. In this case, 5 differentunpurified raw Cannabis leaf wash samples were used to test for affinityand specificity subsequent to the two-step PCR reaction andmicroarray-based hybridization analysis. Both bacterial and fungalanalysis has been performed on all 5 leaf wash samples, by dividing eachsample into halves and subsequently processing them each for analysis ofbacteria or for analysis of fungi, yeast and mold. The data of FIG. 12were obtained by combining the outcome of both assays. FIG. 12 showsthat the combination of two step PCR and microarray hybridizationanalysis, as described in FIG. 9, can be used to analyze the pathogencomplement of a routine Cannabis leaf wash. It is expected, but notshown that such washing of any plant material could be performedsimilarly.

TABLE 12 Representative microarray hybridization data obtained frompurified bacterial DNA standards PURIFIED DNA BACTERIA PANEL AeromonasBacillus Campylobactor hydrophila subtilus ssp. Low Pan Bacteria 443415943 38700 Control Medium Pan Bacteria 7893 33069 28705 Control HighPan Bacteria 14934 23469 32936 Control Low Bile tolerant gram 5364 947867 negative High Bile tolerant gram 55228 339 422 negative TotalColiform 106 101 145 E. coli 104 121 127 E. Coli specific gene 318 255422 E. Coli Stx1 106 116 158 E. Coli Stx2 100 100 126 Enterobacteriacea885 125 211 Salmonella/Enterobacter 115 99 124 Salmonella specific 189175 217 gene Aeromonas 10335 120 123 Pseudomonas 106 107 120 Pseudomonas169 228 173 aeriginosa Xanthomonas 98 188 122 Listeria 117 263 144Campylobacter 148 120 65535 Bacillus Group 2 143 34517 121 E. coli E.coli 0157:H7 Listeria ssp. Low Pan Bacteria 4215 1745 14140 ControlMedium Pan Bacteria 8349 3638 35237 Control High Pan Bacteria 9827 432716726 Control Low Bile tolerant gram 2803 1801 817 negative High Biletolerant gram 24172 14746 1482 negative Total Coliform 8276 9175 139 E.coli 55419 47805 151 E. Coli specific gene 57638 57112 521 E. Coli Stx1134 65535 151 E. Coli Stx2 169 52041 135 Enterobacteriacea 58323 36641179 Salmonella/Enterobacter 190 160 144 Salmonella specific 208 392 212gene Aeromonas 127 139 163 Pseudomonas 130 126 133 Pseudomonas 318 1217208 aeriginosa Xanthomonas 133 143 143 Listeria 136 128 24783Campylobacter 139 153 224 Bacillus Group 2 128 150 137 PseudomonasSalmonella Xanthomonas aeruginosa enterica ssp. Low Pan Bacteria 2643111167 22152 Control Medium Pan Bacteria 39002 17682 24141 Control HighPan Bacteria 38682 28596 22072 Control Low Bile tolerant gram 4852 4453461 negative High Bile tolerant gram 36337 32579 356 negative TotalColiform 145 204 196 E. coli 144 83 147 E. Coli specific gene 695 641461 E. Coli Stx1 142 196 145 E. Coli Stx2 147 117 132 Enterobacteriacea375 23847 204 Salmonella/Enterobacter 138 37520 144 Salmonella specific211 8124 231 gene Aeromonas 142 99 146 Pseudomonas 25866 77 153Pseudomonas 64437 135 424 aeriginosa Xanthomonas 221 80 41903 Listeria144 79 131 Campylobacter 144 88 160 Bacillus Group 2 139 81 134

TABLE 13 Representative microarray hybridization data obtained frompurified bacterial DNA standards PURIFIED DNA FUNGAL PANEL FusariumPenicillium A. fumigatus A. flavus A. niger spp. spp. Mucor Low Pan 42696097 5252 13907 3929 3073 Fungal Control Medium 27006 30445 19746 3097230947 49986 Pan Fungal Control High Pan 64940 64679 54483 47268 6553563932 Fungal Control Negative 119 127 151 107 117 118 control A.fumigatus 62018 232 114 604 126 228 A. flavus 210 65535 116 102 115 128A. niger 113 235 24867 108 115 112 Botrytis 189 205 435 101 126 121Penicillium 171 282 121 100 5891 316 F. solani 112 131 174 16578 113 140Mucor 118 127 113 150 113 29886

FIG. 13 displays representative DNA microarray analysis of anembodiment. In this case, one unpurified (raw) Cannabis leaf wash samplewas used and was compared to data obtained from a commercially-obtainedhomogenous yeast vitroid culture of live Candida to test for affinityand specificity subsequent to the two-step PCR reaction andmicroarray-based hybridization analysis. Both Cannabis leaf wash andcultured fungal analysis have been performed by processing them each foranalysis via probes specific for fungi (see Tables 9 and 11).

The data of FIG. 13 were obtained by combining the outcome of analysisof both the leaf wash and yeast vitroid culture samples. The data ofFIG. 13 show that the combination of two step PCR and microarrayhybridization analysis, as described in FIG. 9, can be used tointerrogate the fungal complement of a routine Cannabis leaf wash asadequately as can be done with a pure (live) fungal sample. It isexpected that fungal analysis via such washing of any plant materialcould be performed similarly.

FIG. 14 shows a graphical representation of the position of PCR primersemployed in a variation of an embodiment for low level detection ofBacteria in the Family Enterobacteriaceae including E. coli. These PCRprimers are used to selectively amplify and dye label DNA from targetedorganisms for analysis via microarray hybridization.

FIGS. 15A-15C illustrate representative DNA microarray analysisdemonstrating assay sensitivity over a range of microbial inputs. Inthis case, certified reference material consisting of enumeratedbacterial colonies of E. coli O157:H7, E. coli O111 (FIGS. 15A-15B) andSalmonella enterica (FIG. 15C) were spiked as a dilution series onto ahops plant surrogate matrix then processed using the assay versiondescribed for FIG. 14. Hybridization results from relevant probes fromFIG. 7 are shown. The larger numbers on the x-axis represents the totalnumber of bacterial colony forming units (CFU) that were spiked ontoeach hops plant sample, whereas the smaller numbers on the x-axisrepresent the number of CFU's of the spiked material that were actuallyinputted into the assay. Only about 1/50 of the original spiked hopssample volume was actually analyzed. The smaller numbers upon the x-axisof FIGS. 15A-15C are exactly 1/50^(th) that of the total (lower) values.As is seen, FIGS. 15A-15C show that the microarray test of an embodimentcan detect less than 1 CFU per microarray assay. The nucleic acidtargets within the bacterial genomes displayed in FIGS. 15A-15C comprise16s rDNA. There are multiple copies of the 16s rDNA gene in each ofthese bacterial organisms, which enables detection at <1 CFU levels.Since a colony forming unit approximates a single bacterium in manycases, the data of FIGS. 15A-15C demonstrate that the present microarrayassay has sensitivity which approaches the ability to detect a single(or a small number) of bacteria per assay. Similar sensitivity isexpected for all bacteria and eukaryotic microbes in that it is knownthat they all present multiple copies of the ribosomal rDNA genes percell.

Tables 14A and 14B show a collection of representative microarrayhybridization data obtained from powdered dry food samples with noenrichment and 18-hour enrichment for comparison. The data shows thatbacterial microbes were successfully detected on the microarrays of thepresent invention without the need for enrichment.

FIG. 16 and Tables 15-17 describes embodiments for the analysis offruit, embodiments for the analysis of vegetables and embodiments forthe analysis of other plant matter. The above teaching shows, byexample, that unprocessed leaf and bud samples in Cannabis and hops maybe washed in an aqueous water solution, to yield a water-wash containingmicrobial pathogens which can then be analyzed via the presentcombination of RSG and microarrays.

If fresh leaf, flower, stem or root materials from fruit and vegetablesare also washed in a water solution in that same way (when fresh, orafter drying and grinding or other types or processing, then the presentcombination of RSG and microarray analysis would be capable ofrecovering and analyzing the DNA complement of those microbes in thoseother plant materials. At least two methods of sample collection arepossible for fruit and vegetables. One method is the simple rinsing ofthe fruit, exactly as described for Cannabis, above. Another method ofsample collection from fruits and vegetables is a “tape pull”, wherein apiece of standard forensic tape is applied to the surface of the fruit,then pulled off. Upon pulling, the tape is then soaked in the standardwash buffer described above, to suspend the microbes attached to thetape. Subsequent to the tape-wash step, all other aspects of theprocessing and analysis (i.e., raw sample genotyping, PCR, thenmicroarray analysis) are exactly as described above.

TABLE 14A Representative microarray data obtained from powdered dry foodsamples Sample Type Whey Protein Whey Protein Chewable Vanilla Pea ShakeVanilla Shake Chocolate Berry Tablet Shake Protein Enrichment time 0 180 18 0 18 0 18 0 18 hours hours hours hours hours hours hours hourshours hours Negative Control Probe 289 318 349 235 327 302 358 325 321299 Total Aerobic Bacteria Probes High sensitivity 26129 38896 1662911901 3686 230 32747 12147 41424 40380 Medium sensitivity 5428 6364 33082794 876 215 7310 2849 15499 8958 Low sensitivity 2044 3419 1471 990 446181 2704 1062 4789 3887 Bile-tolerant Gram-negative Probes Highsensitivity 2639 350 1488 584 307 305 1041 472 15451 8653 Mediumsensitivity 1713 328 892 493 322 362 615 380 6867 4997 Low sensitivity974 600 749 621 595 688 821 929 2459 1662 Total EnterobacteriaceaeProbes High sensitivity 1131 306 363 310 346 318 273 331 4260 3149Medium sensitivity 479 296 320 297 329 339 314 342 1489 990 Lowsensitivity 186 225 203 165 205 181 207 200 216 259 16S rDNA SpeciesProbes Escherichia coli/Shigella spp. 233 205 255 219 207 255 215 214242 198 S. enterica/enterobacter spp. 203 183 186 281 212 299 197 257308 303 Bacillus spp. 154 172 189 114 307 156 169 153 233 259Pseudomonas spp. 549 201 202 251 148 216 303 276 2066 983 OrganismSpecific Gene Probes tuf gene(E. coli) 204 129 180 272 158 190 191 183186 192 stx1 gene(E. coli) 241 178 171 240 289 304 195 245 149 191 stx2gene(E. coli) 145 96 136 125 182 224 130 142 85 127 invA (Salmonellaspp.) 215 265 210 284 204 256 239 285 237 229

TABLE 14B Representative microarray data obtained from powdered dry foodsamples Sample Type Rice Work-out Work-out Vanilla Protein Shake FPShake BR Shake Enrichment time 0 18 0 18 0 18 0 18 hours hours hourshours hours hours hours hours Negative Control Probe 351 351 271 309 299332 246 362 Total Aerobic Bacteria Probes High sensitivity 471 288 17146266 19207 261 41160 47198 Medium sensitivity 161 187 3120 229 3309 31110060 22103 Low sensitivity 186 239 1211 261 1223 264 3673 6750Bile-tolerant Gram-negative Probes High sensitivity 326 372 375 380 412363 1418 358 Medium sensitivity 304 362 341 391 308 356 699 394 Lowsensitivity 683 942 856 689 698 864 848 665 Total EnterobacteriaceaeProbes High sensitivity 277 329 317 327 298 326 290 349 Mediumsensitivity 326 272 296 291 297 263 262 307 Low sensitivity 215 207 204288 213 269 195 247 16S rDNA Species Probes Escherichia coli/Shigellaspp. 228 229 216 267 221 253 220 207 S. enterica/enterobacter spp. 226281 238 268 197 254 255 216 Bacillus spp. 157 166 812 208 915 216 415168 Pseudomonas spp. 199 225 247 251 211 259 277 225 Organism SpecificGene Probes tuf gene(E. coli) 150 149 126 206 163 212 215 166 stx1gene(E. coli) 270 247 211 299 239 307 175 185 stx2 gene(E. coli) 158 178127 205 138 175 128 100 invA (Salmonella spp.) 257 241 249 264 220 258239 245

The data of Tables 15-17 demonstrates that simple washing of the fruitand tape pull sampling of the fruit generate similar microbial data. Theblueberry sample is shown to be positive for Botrytis, as expected,since Botrytis is a well-known fungal contaminant on blueberries. Thelemon sample is shown to be positive for Penicillium, as expected, sincePenicillium is a well-known fungal contaminant for lemons.

TABLE 15 Representative microarray hybridization data obtained fromblueberry and lemon washes. Sample Blueberry Lemon Collection TypeProduce Wash Protocol Wash 1 blueberry in 2 ml Wash 1 piece moldy 20 mMBorate, vortex 30 lemon in 2 ml 20 mM seconds Borate, vortex 30 secondsDilution Factor NONE 1:20 NONE 1:20 A. fumigatus 1 65 61 62 57 A.fumigatus 2 66 61 58 131 A. fumigatus 3 69 78 55 127 A. fumigatus 4 80198 63 161 A. fumigatus 5 98 68 59 70 A. flavus 1 111 65 197 58 A.flavus 2 64 66 71 49 A. flavus 3 72 79 54 49 A. flavus 4 95 71 66 125 A.flavus 5 59 55 45 47 A. niger 1 91 75 61 61 A. niger 2 185 68 61 57 A.niger 3 93 66 62 61 A. niger 4 1134 74 75 64 Botrytis spp. 1 26671 2760560 55 Botrytis spp. 2 26668 35611 59 57 Penicillium spp. 1 63 69 24444236 Penicillium spp. 2 71 69 4105 7426 Fusarium spp. 1 175 69 59 78Fusarium spp. 2 71 73 84 62 Mucor spp. 1 71 57 58 61 Mucor spp. 2 61 29066 61 Total Yeast and Mold 1 20052 21412 8734 7335 Total Yeast and Mold2 17626 8454 5509 5030

TABLE 16 Representative microarray hybridization data obtained fromblueberry washes and tape pulls Sample Moldy Blueberry Collection TypeTape Pull ID 1A1 1A1 1A2 1A2 1A3 1A3 1B1 1B1 1B2 1B2 1B3 1B3 CollectionPoint 1 500 ul 20 mM Borate Buffer, vortex 30 seconds 500 ul 20 mMBorate + Triton Buffer, vortex 30 seconds Collection Point 2 Add 15 mgzirconia beads, Add 15 mg zirconia beads, vortex, Heat 5 min 95° C.,vortex, Heat 5 min 95° C., Vortex 15 seconds Vortex 15 secondsCollection Point 3 Heat 5 min Heat 5 min 95° C. vortex 95° C. vortex 15seconds 15 seconds Dilution Factor NO 1:20 NO 1:20 NO 1:20 NO 1:20 NO1:20 NO 1:20 A. fumigatus 1 66 388 83 77 97 313 95 68 76 55 75 60 A.fumigatus 2 97 100 82 118 69 56 87 67 185 76 58 52 A. fumigatus 3 77 9482 1083 87 61 93 84 75 378 73 64 A. fumigatus 4 84 151 94 118 96 80 11585 85 93 190 88 A. fumigatus 5 63 75 96 71 78 61 98 74 68 98 70 533 A.flavus 1 200 107 113 61 204 58 105 73 62 68 64 65 A. flavus 2 70 104 6457 133 281 111 78 377 314 57 50 A. flavus 3 83 90 94 150 99 90 96 2221162 86 80 73 A. flavus 4 76 125 92 146 87 174 241 78 115 69 105 85 A.flavus 5 80 153 77 72 78 439 71 86 280 58 62 57 A. niger 1 409 178 12272 80 70 76 71 152 117 65 53 A. niger 2 78 292 79 65 715 666 74 70 68731 70 54 A. niger 3 86 76 87 558 78 60 70 81 96 63 478 58 A. niger 4164 70 92 108 197 69 130 75 76 148 73 65 Botrytis spp. 1 41904 2654928181 29354 25304 25685 57424 33783 57486 49803 33176 32153 Botrytisspp. 2 36275 25518 29222 27076 26678 27675 49480 32899 52817 34322 2969332026 Penicillium spp. 1 80 81 83 64 96 60 79 80 176 60 385 53Penicillium spp. 2 90 93 81 80 114 59 98 69 470 65 478 56 Fusarium spp.1 77 71 69 62 112 55 61 274 617 81 59 757 Fusarium spp. 2 91 82 107 74101 65 91 66 123 63 71 583 Mucor spp. 1 90 314 73 88 105 61 77 79 741180 172 74 Mucor spp. 2 83 69 73 69 91 67 111 102 455 88 70 133 Total Y& M 1 23637 18532 15213 17668 18068 19762 18784 15550 20625 17525 2581318269 Total Y & M 2 12410 8249 9281 11526 8543 13007 14180 14394 99058972 15112 12678

The data embodied in FIG. 16 and Tables 15-17 demonstrate the use of anembodiment, for the recovery and analysis of yeast microbes on thesurface of fruit (blueberries and lemons in this case), but anembodiment could be extended to other fruits and vegetables for theanalysis of both bacterial and fungal contamination.

TABLE 17 Representative microarray hybridization data obtained fromlemon washes and tape pulls. Sample Moldy Lemon Collection Type TapePull ID 1A1 1A2 1A3 1B1 1B2 Lemon Lemon Lemon Lemon Lemon CollectionPoint 1 500 ul 20 mM Borate + Triton Buffer, vortex 30 secondsCollection Point 2 Add 15 mg Add 15 mg zirconia beads, zirconia beads,vortex, Heat 5 min vortex, Heat 5 min 95° C., Vortex 95° C., Vortex 15seconds 15 seconds Collection Point 3 Heat 5 min 95° C. vortex 15seconds Dilution Factor NONE A. fumigatus 1 96 83 75 83 64 A. fumigatus2 221 73 71 66 101 A. fumigatus 3 87 88 85 92 122 A. fumigatus 4 83 8591 72 97 A. fumigatus 5 448 100 84 114 78 A. flavus 1 85 79 70 66 63 A.flavus 2 77 82 77 79 63 A. flavus 3 133 66 86 60 67 A. flavus 4 96 85 8198 88 A. flavus 5 68 62 65 106 59 A. niger 1 73 88 77 73 73 A. niger 274 84 81 71 103 A. niger 3 90 86 87 74 78 A. niger 4 82 93 104 86 161Botrytis spp. 1 82 75 75 77 68 Botrytis spp. 2 91 74 83 67 62Penicillium spp. 1 3824 5461 5500 4582 5290 Penicillium spp. 2 7586 838011177 6528 8167 Fusarium spp. 1 101 62 61 70 279 Fusarium spp. 2 77 12278 68 233 Mucor spp. 1 74 110 89 76 57 Mucor spp. 2 132 1302 90 84 61Total Yeast and Mold 1 8448 12511 9249 12844 8593 Total Yeast and Mold 29275 8716 11585 10758 4444

Table 18 shows embodiments for the analysis of environmental watersamples/specimens. The above teaching shows by example that unprocessedleaf and bud samples in Cannabis and hops may be washed in an aqueouswater solution, to yield a water-wash containing microbial pathogenswhich can then be analyzed via the present combination of Raw SampleGenotyping (RSG) and microarrays. If a water sample containing microbeswere obtained from environmental sources (such as well water, or seawater, or soil runoff or the water from a community water supply) andthen analyzed directly, or after ordinary water filtration toconcentrate the microbial complement onto the surface of the filter,that the present combination of RSG and microarray analysis would becapable of recovering and analyzing the DNA complement of thosemicrobes.

TABLE 18 Representative microarray data from raw water filtrate. SampleID Negative 2 H 2 H 9 D 9 D 21 21 23 23 25 25 Control Imager CalibrationHigh 311 335 322 379 341 348 345 325 354 343 333 Imager Calibration Med280 314 268 286 288 231 253 295 267 295 244 Imager Calibration Low 245296 302 324 254 268 293 285 271 340 275 Cannabis cont. 310 330 313 255323 368 313 322 274 332 322 Cannabis cont. 313 237 298 271 298 288 296280 249 284 297 Cannabis cont. 208 265 276 250 267 327 255 258 253 282370 Total Yeast and Mold 284 324 290 307 272 361 296 288 271 321 469Total Yeast and Mold 251 259 294 290 309 308 285 281 275 299 293 TotalYeast and Mold 282 280 294 280 299 284 275 286 299 259 232 Total Aerobicbacteria High 40101 42007 47844 47680 45102 44041 43520 41901 4645946783 135 Total Aerobic bacteria Medium 14487 12314 24189 26158 1971216210 17943 15474 25524 18507 157 Total Aerobic bacteria Low 4885 56297625 6456 5807 4505 5316 6022 6264 6974 159 Negative Control 293 359 303339 312 329 306 377 307 335 307 Aspergillus fumigatus 285 291 284 268289 265 271 281 269 248 228 Aspergillus flavus 184 211 201 344 237 179212 213 163 204 171 Aspergillus niger 226 213 228 273 190 195 245 206222 209 172 Botrytis spp. 219 285 258 302 275 219 202 288 221 248 214Alternaria spp. 81 97 76 89 58 76 75 175 117 174 167 Penicillium paxilli135 162 215 142 127 161 103 115 238 190 200 Penicillium oxalicum 119 107161 131 135 241 178 158 140 143 194 Penicillium spp. 50 123 179 177 128138 146 163 148 115 184 Can. alb/trop/dub 261 236 235 230 250 213 276244 245 237 194 Can. glab/Sach & Kluv spp. 146 165 196 128 160 215 185217 215 177 225 Podosphaera spp. 111 119 100 122 192 105 95 43 169 27143 Bile-tolerant Gram-negative High 16026 9203 13309 8426 16287 1411610557 17558 15343 14285 183 Bile-tolerant Gram-negative Medium 1230211976 9259 10408 13055 10957 11242 8416 9322 11785 196 Bile-tolerantGram-negative Low 5210 7921 3818 3984 7224 6480 4817 6933 5021 5844 240Total Enterobacteriaceae High 193 248 389 357 215 214 198 220 276 208210 Total Enterobacteriaceae Med 246 214 297 246 244 224 219 245 252 229207 Total Enterobacteriaceae Low 165 140 158 119 151 180 150 167 182 174132 Total Coliform 121 148 158 117 129 117 155 157 125 178 152Escherichia coli specific gene 31821 115 132 155 127 62 86 121 59 90 234stx1 gene 67 0 2 0 0 23 21 28 0 0 116 stx2 gene 17 36 174 0 61 47 0 5133 0 85 Salmonella specific gene 181 172 245 172 178 212 157 243 174 156146 Bacillus spp. 137 135 174 112 164 143 163 182 168 152 149Pseudomonas spp. 271 74 332 56 366 133 91 114 60 179 555 Escherichiacoli/Shigella spp. 103 124 221 124 90 144 130 121 137 143 158 Salmonellaenterica/enterobacter spp. 124 98 131 119 136 88 121 77 128 140 124Erysiphe Group 2 278 221 237 230 245 254 250 220 205 236 233 Trichodermaspp. 105 157 204 152 180 154 130 161 201 180 150 Escherichia coli 429431 551 576 549 406 407 484 556 551 293 Aspergillus niger 218 212 216297 255 312 221 202 238 231 209 Escherichia coli/Shigella spp. 163 193220 202 308 280 121 271 341 317 124 Aspergillus fumigatus 713 865 862830 784 657 827 803 746 812 793 Aspergillus flavus 155 261 198 156 239171 250 218 210 258 219 Salmonella enterica 136 98 85 43 109 47 23 12370 100 135 Salmonella enterica 68 53 52 41 60 92 26 28 55 81 116

The data embodied in Table 18 were obtained from 5 well-water samples(named 2H, 9D, 21, 23, 25) along with 2 samples of milliQ laboratorywater (obtained via reverse osmosis) referred to as “Negative Control”.All samples were subjected to filtration on a sterile 0.4 um filter.Subsequent to filtration, the filters, with any microbial contaminationthat they may have captured, were then washed with the standard washsolution, exactly as described above for the washing of Cannabis andfruit. Subsequent to that washing, the suspended microbes in washsolution were then subjected to exactly the same combination ofcentrifugation (to yield a microbial pellet) then lysis and PCR of theunprocessed pellet-lysate (exactly as described above for Cannabis),followed by PCR and microarray analysis, also as described for Cannabis.

The data seen in Table 18 demonstrate that microbes collected onfiltrates of environmental water samples can be analyzed via the samecombination of raw sample genotyping, then PCR and microarray analysisused for Cannabis and fruit washes. The italicized elements of Table 18demonstrate that the 5 unprocessed well-water samples all containaerobic bacteria and bile tolerant gram-negative bacteria. The presenceof both classes of bacteria is expected for unprocessed (raw) wellwater. Thus, the data of Table 18 demonstrate that this embodiment ofthe present invention can be used for the analysis of environmentallyderived water samples.

The above teaching shows that unprocessed leaf and bud samples inCannabis and hops may be washed in an aqueous water solution to yield awater-wash containing microbial pathogens which can then be analyzed viathe present combination of RSG and microarrays. The above data also showthat environmentally-derived well water samples may be analyzed by anembodiment. Further, if a water sample containing microbes were obtainedfrom industrial processing sources (such as the water effluent from theprocessing of fruit, vegetables, grain, meat) and then analyzeddirectly, or after ordinary water filtration to concentrate themicrobial complement onto the surface of the filter, that the presentcombination of RSG and microarray analysis would be capable ofrecovering and analyzing the DNA complement of those microbes.

Further, if an air sample containing microbes as an aerosol or adsorbedto airborne dust were obtained by air filtration onto an ordinaryair-filter (such as used in the filtration of air in an agricultural orfood processing plant, or on factory floor, or in a public building or aprivate home) that such air-filters could then be washed with a watersolution, as has been demonstrated for plant matter, to yield amicrobe-containing filter eluate, such that the present combination ofRaw Sample Genotyping (RSG) and microarray analysis would be capable ofrecovering and analyzing the DNA complement of those microbes.

While the foregoing written description of an embodiments enables one ofordinary skill to make and use what is considered presently to be thebest mode thereof, those of ordinary skill will understand andappreciate the existence of variations, combinations, and equivalents ofthe specific embodiment, method, and examples herein. The presentdisclosure should therefore not be limited by the above describedembodiments, methods, and examples, but by all embodiments and methodswithin the scope and spirit of the present disclosure.

Example 4

Method of Using the 3-Dimensional Lattice Microarray System forQuantitative DNA Analysis.

Sample Processing

Harvesting Microbes from plant surface comprises the following steps:

1. Wash the plant sample or tape pull in 1× phosphate buffered saline(PBS);

2. Remove plant material/tape;

3. Centrifuge to pellet cells and iscard supernatant;

3. Resuspend in PathogenDx® Sample Prep Buffer pre-mixed with SampleDigestion Buffer;

3. Heat at 55° C. for 45 minutes;

3. Vortex to dissipate the pellet;

3. Heat at 95° C. for 15 minutes; and

3. Vortex and centrifuge briefly to obtain a sample comprising DNA fromone or more types of microbes.

Addition of a Synthetic DNA to the Processed Sample

A known amount (known copy number) of synthetic DNA is added to thesample obtained in the sample processing step described above. Thesynthetic DNA has a length and sequence structure similar to that of the16s (bacteria) or ITS2 (eukaryote) DNA sequence being amplified, butwith a central region sequence that is distinct, to distinguish it frombacterial and eukaryotic DNA in the sample. The 5′ and 3′ end sequencesof the synthetic DNA are designed to be substantially identical to aconsensus sequence in the unknown bacterial or unknown eukaryotic DNAbeing queried, to allow amplification using the same pair of PCR primersused for amplification of the unknown DNA in the sample. Examples ofsuch consensus sequences are shown in SEQ ID NO: 152 and SEQ ID NO: 153(Table 19). These features allow unbiased amplification of both thesynthetic DNA and the unknown microbial pool DNA in the sample. Examplesof synthetic DNA sequences are shown in SEQ ID NO: 154 to SEQ ID NO: 157(Table 19).

Amplification by PCR

The sample comprising the synthetic DNA sequence was amplified (PCRReaction #1) using locus specific primer pairs (Tables 6 and 20). Theproduct of PCR Reaction #1 (14) was then subjected to a second PCRreaction (PCR Reaction #2) using a pair of labeling primers (Tables 6and 20), which additionally amplified and labeled the two targetedregions to generate fluorophore labeled amplicons. The product of thePCR Reaction #2 (504) was then diluted 1-1 with hybridization buffer(4×SSC+5×Denhardt's solution) and then applied directly to themicroarray for hybridization.

Hybridization

Because the prior art method of microarray manufacture allows DNA to beanalyzed via hybridization without the need for pre-treatment of themicroarray surface, the use of the microarray is simple, and involves 6manual or automated

1. Pipette the amplified DNA+binding buffer onto the microarray to whichare immobilized oligonucleotide probe sequence for the pathogen genebeing queried and the synthetic DNA used as internal reference standard(Tables 7-11 and 21).

2. Incubate for 30 minutes to allow DNA binding to the microarray(typically at room temperature, RT).

3. Remove the DNA+binding buffer by pipetting

4. Pipette 50 uL of wash buffer onto the microarray(0.4×SSC+0.5×Denhardt's) and incubate 5 min at RT.

5. Remove the wash buffer by pipetting.

6) Repeat steps 4 and 5

7) Perform image analysis at 532 nm and 635 nm to detect the probe spotlocation (532 nm) and PCR product hybridization (635 nm).

TABLE 19 Concensus sequences and Synthetic DNA sequences SEQ ID NO: 152Consensus sequence corresponding to the ITS2 domain in eukaryotesincluding yeast, mold fungi (TYM Quant Control)NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACTTYCAACAAYGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGTGAATCATCGARTCTTTGAACGCANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGCATATCAATAAGCGGAGGAAAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN SEQ ID NO: 153Consensus sequence corresponding to the rDNA domain in prokaryotesNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCACAYTGGRACTGAGACACGGNNNNNNCTCCTACGGGAGGCAGCAGTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAYSSAGCMAYGCCGCGTGDRBGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNATTGACGTTACCCGCNNNNNNNNNNNNNNNNNNNNNNNTGCCAGCAGCCGCGGTAATACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNSEQ ID NO: 154 Synthetic DNA region compatible for use as an internalreference standard with the ITS2 domain in eukaryotesTACTATTCAGCCTCTGTACGTGCTTCATGTAAATTGAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGACTTGCGATAAGTAATGTGAATTGCAGAATTCAGTGCATCATAGAAACTATGTACGCAAATTGCGCCCCTTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCTTAGCTTGGTATTGAGTCTATGTCAGTAATGGCAGGCTCTAAAATCAGTGGCGGCGCCGCTGGGTCCTGAACGTAGTAATATTTCTTGTCACCGTTTCTAGGTGTGCTTCTGTCTATACCCAAATTCTTCTATGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAAAGCACGCCGTCTAG AAGCACGATCAGAGGCTGAATACTASEQ ID NO: 155 Synthetic DNA region compatible for use as an internalreference standard with the rDNA domain in prokaryotesTACTATTCAGCCTCTGTACGTGCTTCATGTAAATTGACACACTGGAACTGAGACACGGTCCAGACTCCATCGGGAGCGAGCATGGGGGAATATTGCACAATGGGCGCAAGCCTGATGGACCCTAGCCGCCACTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAGGGAATGAAAGTATATACCTTTCGTCATGTACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGCACGCCGTCTAGAAGCACGATCAGAG GCTGAATACTASEQ ID NO: 156 Synthetic DNA region compatible for use as an internalreference standard with the rDNA domain in prokaryotesincluding Bile-tolerant Gram- negative bacteriaTACTATTCAGCCTCTGTACGTGCTTCATGTAAATTGACACACTGGAACTGAGACACGGTCCAGACTCCTGCAGGAGACGGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGTATCCCTGACGCAGATATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGTCGTAAAACTAATACACTTGCTGTTTGAACTTACCCAGAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGCACGCCGTCTAGAAGCACGATCAGAG GCTGAATACTASEQ ID NO: 157 Synthetic DNA region compatible for use as an internalreference standard with the rDNA domain prokaryotes includingthe Enterobacteriaceae FamilyTACTATTCAGCCTCTGTACGTGCTTCATGTAAATTGACACACTGGAACTGAGACACGGTCCAGACTCCTAGCGGAGCGAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGACGCCAGTCCGCTGGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGAGGTAAATGTAATACTCTTGCTACTTGAGCTTACCCCGAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGCACGCCGTCTAGAAGCACGATCAGAG GCTGAATACTA

TABLE 20 Primers used for Locus PCR andLabeling PCR Amplification Reactions SEQ ID NO. Primer targetPrimer sequence SEQ ID NO: 133 Universal primers TTCAGCCTCTGTAfor preamplification CGTGCTTCATG of Fungal and Bacterial controlfragments SEQ ID NO: 134 Universal primers TTCAGCCTCTGATfor preamplification CGTGCTTCTAG of Fungal and Bacterial controlfragments SEQ ID NO: 135 Fungal RSG Primers TTTACTTTCAACA (All Fungus)AYGGATCTCTTGG SEQ ID NO: 136 Fungal RSG Primers CTTTTCCTCCGCT(All Fungus) TATTGATATG SEQ ID NO: 137 Bacterial RSG  TTTCACACTGGRAPrimers CTGAGACACG (All Bacteria) SEQ ID NO: 138 Bacterial RSG TTTTGTATTACCG Primers CGGCTGCTGGC (All Bacteria) SEQ ID NO: 139Fungal Labeling TTTGCATCGATGA Primers(All Fungus) AGAACGCAGCSEQ ID NO: 140 Fungal Labeling TTTTCCTCCGCTT Primers(All Fungus)ATTGATATGC SEQ ID NO: 141 Bacterial Labeling TTTCACACTGGRA PrimersCTGAGACACGG SEQ ID NO: 30 Bacterial Labeling TTTTGTATTACCG PrimersCGGCTGCTGGCA

TABLE 21 Oligonucleotide probe sequences SEQ ID NO: 132 Negative controlTTTTTTCTACTACCTATGCTGATTCACTCT TTTT SEQ ID NO: 37 Total Aerobic bacteriaTTTTTTTTTCCTACGGGAGGCAGTTTTTTT (High) SEQ ID NO: 38Total Aerobic bacteria TTTTTTTTCCCTACGGGAGGCATTTTTTTT (Medium)SEQ ID NO: 39 Total Aerobic bacteria TTTATTTTCCCTACGGGAGGCTTTTATTTT(Low) SEQ ID NO: 47 Bile-tolerant Gram- TTTTTCTATGCAGTCATGCTGTGTGTRTGTnegative (High) CTTTTT SEQ ID NO: 48 Bile-tolerant Gram-TTTTTCTATGCAGCCATGCTGTGTGTRTTT negative (Medium) TTTT SEQ ID NO: 49Bile-tolerant Gram- TTTTTCTATGCAGTCATGCTGCGTGTRTTT negative (Low) TTTTSEQ ID NO: 54 Coliform/Entero- TTTTTTCTATTGACGTTACCCGCTTTTTTTbacteriaceae SEQ ID NO: 41 EnterobacteriaceaeTTTTTTCTATTGACGTTACCCGTTTTTTTT (Medium sensitivity) SEQ ID NO: 40Enterobacteriaceae TTTATTCTATTGACGTTACCCATTTATTTT (Low sensitivity)SEQ ID NO: 86 Total Yeast and Mold TTTTTTTTGAATCATCGARTCTTTGAACGC(High sensitivity) ATTTTTTT SEQ ID NO: 88 Total Yeast and MoldTTTTTTTTGAATCATCGARTCTTTGAACGT (Medium sensitivity) TTTTTT SEQ ID NO: 87Total Yeast and Mold TTTTTTTTGAATCATCGARTCTCCTTTTTTT (Low sensitivity)SEQ ID NO: 142 Total Aerobic Bacteria TTTTTTTTTCCATCGGGAGCGAGTTTTTTTQuantitative Control (internal reference standard) SEQ ID NO: 143Bile-tolerant Gram- TTTTTCTATGTATTCCTGATGTAGATRTGT negative QuantitativeCTTTTT Control (internal reference standard) SEQ ID NO: 144Enterobacteriaceae/ TTTTTTCTCTTGAGCTTACCCCGTTTTTTT Coliform QuantitativeControl (internal reference standard) SEQ ID NO: 145Total Yeast and Mold TTTTTTTTGCATCATAGAAACTTTGTACGC Quantitative ControlATTTTTTT (internal reference standard) SEQ ID NO: 103Candida spp. Group 2 TTTTACTGTTTGGTAATGAGTGATACTCTC ATTTT SEQ ID NO: 146Golovinomyces spp. TTTATTTAATCAATCCATCATCTCAAGTCT TTTT SEQ ID NO: 108Fusarium spp TTTTTTTTAACACCTCGCRACTGGAGATTT TTTT SEQ ID NO: 107Fusarium oxysporum TTTTTTTCTCGTTACTGGTAATCGTCGTTT TTTT SEQ ID NO: 42Escherichia coli/ TTTTCTAATACCTTTGCTCATTGACTCTTT Shigella 1SEQ ID NO: 74 Salmonella TTTTTTTGTTGTGGTTAATAACCGATTTTTenterica/Enterobacter 1 SEQ ID NO: 72 Pseudomonas spp. 3TTTATTTTAAGCACTTTAAGTTGGGATTTT ATTT SEQ ID NO: 78 Staphylococcus aureusTTTATTTTCATATGTGTAAGTAACTGTTTTA 1 TTT SEQ ID NO: 147 Listeria spp.TTTATTTTGATAAGAGTAACTGCTTGCTTT ATTT SEQ ID NO: 45 Bacillus spp. Group1TTTTTCAGTTGAATAAGCTGGCACTCTTTT SEQ ID NO: 46 Bacillus spp. Group2TTTTTTCAAGTACCGTTCGAATAGTTTTTT SEQ ID NO: 90 Aspergillus flavus 1TTTTTTCGCAAATCAATCTTTTTCCAGTCT TTTT SEQ ID NO: 92Aspergillus fumigatus 1 TTTCTTTTCGACACCCAACTTTATTTCCTT ATTTSEQ ID NO: 95 Aspergillus niger 1 TTTTTTCGACGTTTTCCAACCATTTCTTTTSEQ ID NO: 100 Botrytis spp TTTTTTTCATCTCTCGTTACAGGTTCTCGG TTCTTTTTTTSEQ ID NO: 116 Penicillium oxalicum TTTTTTACACCATCAATCTTAACCAGGCCT TTTTSEQ ID NO: 117 Penicillium paxilli TTTTTTCCCCTCAATCTTTAACCAGGCCTT TTTTSEQ ID NO: 118 Penicillium spp TTTTTTCAACCCAAATTTTTATCCAGGCCT TTTTSEQ ID NO: 89 Alternaria spp TTTTTTCAAAGGTCTAGCATCCATTAAGTT TTTTSEQ ID NO: 101 Candida albicans TTTTTTTTTGAAAGACGGTAGTGGTAAGTT TTTTSEQ ID NO: 105 Cladosporium spp TTTTTTTTGTGGAAACTATTCGCTAAAGTT TTTTSEQ ID NO: 99 Blumeria TTTATTTGCCAAAAMTCCTTAATTGCTCTT TTTTSEQ ID NO: 148 Mucor spp. TTTTTTCTCCAVVTGAGYACGCCTGTTTCA GTATCTTTTTTSEQ ID NO: 120 Podosphaera spp TTTTTTGACCTGCCAAAACCCACATACCAT TTTTSEQ ID NO: 149 Saccharomyces spp. TTTATCTTAGGCGAACAATGTTCTTAAATC TTTTSEQ ID NO: 150 Aspergillus terreus TTTTTTACGCATTTATTTGCAACTTGCCTT TTTTSEQ ID NO: 151 Podosphaera spp. TTTTTCGTCCCCTAAACATAGTGGCTTTTTImage Analysis

Image Analysis was performed at two wavelengths (532 nm and 635 nm) on araster-based confocal scanner: GenePix 4000B Microarray Scanner, withthe following imaging conditions: 33% Laser power, 400 PMT setting at532 nm/33% Laser Power, 700 PMT setting at 635 nm. FIG. 3 shows anexample of the structure and hybridization performance of themicroarray.

FIG. 3A reveals imaging of the representative microarray, describedabove, after hybridization and washing, as visualized at 635 nm. The 635nm image is derived from signals from the (red) CY5 fluor attached tothe 5′ terminus of the bifunctional polymer linker OligodT which hadbeen introduced during microarray fabrication as a positional marker ineach microarray spot (see FIG. 1 and Table 3). The data in FIG. 3Aconfirm that the CY5-labelled OligodT has been permanently linked to themicroarray surface, via the combined activity of the bi-functionallinker and subsequent UV-crosslinking, as described in FIG. 1.

FIG. 3B reveals imaging of the representative microarray described aboveafter hybridization and washing as visualized at 532 nm. The 532 nmimage is derived from signals from the (green) CY3 fluor attached to the5′ terminus of PCR amplified DNA obtained during PCR Reaction #2. It isclear from FIG. 3B that only a small subset of the 48 discrete probesbind to the CY3-labelled PCR product, thus confirming that the presentmethod of linking nucleic acid probes to form a microarray (FIG. 1)yields a microarray product capable of sequence specific binding to a(cognate) solution state target. The data in FIG. 3B reveal theunderlying 3-fold repeat of the data (i.e., the array is the same set of48 probes printed three times as 3 distinct sub-arrays to form the final48×3=144 element microarray. The observation that the same set of 48probes can be printed 3-times, as three repeated sub-domains show thatthe present invention generates microarray product that is reproducible.

FIG. 3C reveals imaging of the representative microarray, describedabove, after hybridization and washing, as visualized with both the 532nm and 635 nm images superimposed. The superimposed images display theutility of parallel attachment of a CY5-labelled OligodT positionalmarker relative to the sequence specific binding of the CY3-labelled PCRproduct.

Example 5

Quantitation of Absolute DNA Copy Number

Quantitation of absolute DNA copy number for a microbe of interest in asample as disclosed in the present invention is achieved by introducinginto the sample, a known copy number of a synthetic DNA before the firstPCR amplification step. The synthetic DNA has a length and sequencestructure similar to that of the 16s (bacteria) or ITS2 (eukaryote) DNAamplicons generated in the first PCR (FIGS. 17A-17B and 18), but with acentral region sequence that is distinct, to distinguish it frombacterial and eukaryotic DNA in the sample. The 5′ and 3′ end sequencesof the synthetic DNA are designed to be substantially identical to aconsensus sequence in the unknown bacterial or unknown eukaryotic DNAbeing queried, to allow amplification using the same pair of PCR primersused for amplification of the unknown DNA in the sample. These featuresallow the synthetic DNA to be amplified by the same pair of PCR primersused to amplify the hypervariable region of the unknown microbes whichare being queried. As a result, the synthetic DNA is indistinguishablefrom the microbial pool DNA in the sample in terms of primer/templatehybridization during the first and second PCR amplification reactions.This ensures unbiased amplification of the synthetic (internal referencestandard) and microbial (unknown) DNA, such that signal intensity foramplicons from the queried DNA and synthetic DNA are proportional to theoriginal copy number of each DNA respectively (discussed below).Examples of such consensus sequences are shown in SEQ ID NO: 152 and SEQID NO: 153 (Table 19). Examples of synthetic DNA sequences are shown inSEQ ID NO: 154 to SEQ ID NO: 157 (Table 19).

PCR, as deployed in the present microarray-based analysis is generallyperformed as a type of end-point PCR. Although in its simplestapproximation, the polymerase chain reaction (PCR) has been described asa type of chain reaction because in the beginning of the PCR reactionprocess, when the DNA target strands are extremely dilute, each cycle ofPCR will, in general lead to a geometric increase in product DNA, nearlyexactly a 2-fold increase in amplified DNA product for each new PCRthermal cycle. If such a 2-fold increase were to occur indefinitely, theconcentration of amplified DNA product should theoretically increaseexponentially to infinity. In practice however, such a limitless DNAproduct increase does not occur, and one observes the well-known“S-shaped” sigmoidal PCR response curve wherein one or more componentsof the PCR reactant mixture or, one or more components of the PCRproduct mixture leads to a saturation in amplicon production. It isgenerally believed that such “leveled-off” S-shaped PCR curves occursdue to consumption of the PCR primers in the reaction mixture.Therefore, in the present invention, the concentration of PCR primers inboth of the PCR reactions (first, locus PCR and second, labeling PCR,see FIGS. 17A, 17B and 18) is intentionally kept at a large molarexcess, so that primer depletion cannot contribute to saturation of PCRamplification. However, even with an excess of PCR reactants, it wasobserved that amplicon generated from both PCRs continue to “level-off”to produce a complex “end-point” PCR wherein the number of ampliconswill no longer increase despite increasing concentration of thereactants. This saturation in response occurs due to enzymaticend-product inhibition. In the present invention, such inhibition isenabled to generate a substantial amount of PCR product, so as to ensurethat microarray hybridization remains highly selective and sensitive tolow microbial density in the sample.

The key to the present invention is therefore based upon deployment ofthe above-mentioned synthetic DNA internal reference standards so thateven as the PCR reaction saturates by means of end-product inhibition(or any other mechanism of PCR leveling) the relative abundance of anyDNA derived from an unknown amount of its microbial source, can bedirectly compared to that of the internal reference standard, so thatthe abundance of the unknown DNA, relative to that of the knownsynthetic DNA (internal reference standard) becomes insensitive to theextent of PCR reaction. This effect was exploited in this invention withthe understanding that as the PCR reaction approaches saturation(associated with end-product inhibition), the amounts of variousamplified species begin to interact as discussed below:

i) Unknown DNA Copies=Standard DNA Copies

When the number of copies of the synthetic DNA (standard species) isequal to the number of copies of an microbial DNA (unknown species) inthe original sample, the ratio of microbial DNA to synthetic DNA will beequal to 1 from the beginning of the PCR reaction (where amplificationis exponential) to the end of the PCR reaction (where amplificationbegins to saturate via end-product inhibition).C _(n) /C _(o) =S _(n) /S _(o) where,C_(n)=the number of microbial DNA copies of each type (n) present in theoriginal sample mixture added to the first of two tandem PCR reactionsused to prepare amplicons for microarray analysis,C_(o)=the number of known synthetic DNA copies (internal referencestandard) added to the first of two PCR reactions used to prepareamplicons for microarray analysis,S_(n)=relative fluorescence units (RFU) signal data obtained after PCRamplification, and microarray hybridization of the nth microbialspecies, followed by image analysis,S_(o)=relative fluorescence units (RFU) signal data obtained after PCRamplification, and microarray hybridization of the synthetic DNAspecies, followed by image analysis,

ii) Unknown DNA Copies>Standard DNA Copies

When the microbial DNA (unknown species) were in large excess over theknown synthetic DNA (standard species), the ratio of microbial DNA tosynthetic DNA will be >1. In this situation, at the beginning of the PCRreaction, the relative abundance of amplicons (PCR product) will be atruthful representation of the original input strand ratio(unknown:standard). As the reaction proceeds towards saturation however,the more abundant species (unknown species in this case) will approachend-product saturation first, either due to consumption of one or morereactants or due to enzyme inhibition by PCR reactionproducts—pyrophosphate and/or amplicons. As a result, amplification ofthe relatively less abundant standard species will be inhibited. In thissituation, abundance of amplified DNA product species will retain thecorrect qualitative relationship of “unknown species>standard species”,whereas the ratio of DNA products will no longer be linearly related tothe unknown:standard ratio in the original input sample.C _(n) /C _(o) ≠S _(n) /S _(o)

Such a non-linear relationship between input and response is well knownin chemical and physical analysis such as the relationship between graindensity versus photon exposure in chemical film or in a charge-coupleddevice (CCD) where, the relative brightness of photographic inputs aremaintained in relative rank order in the resulting image, yet a detailedunderstanding of the non-linear response is required to predict relativeabundance of the original inputs (analogous to DNA copy number in theoriginal sample) from the response data (analogous to PCR product signalafter amplification and microarray hybridization).

iii) Unknown DNA Copies<Standard DNA Copies

When the microbial DNA (unknown species) were less than the knownsynthetic DNA (standard species), the ratio of microbial DNA tosynthetic DNA will be <1. In this situation, at the beginning of the PCRreaction, the relative abundance of amplicons (PCR product) will be atruthful representation of the original input strand ratio(unknown:standard). As the reaction proceeds towards saturation however,the more abundant species (standard species in this case) will approachend-product saturation first, either due to consumption of one or morereactants or due to enzyme inhibition by PCR reactionproducts—pyrophosphate and/or amplicons. As a result, amplification ofthe relatively less abundant unknown species will be inhibited. In thissituation, abundance of amplified DNA product species will retain thecorrect qualitative relationship of “unknown species>standard species”,whereas the ratio of DNA products. will no longer be linearly related tothe unknown:standard ratio in the original input sample;C _(n) /C _(o) ≠S _(n) /S _(o)

Such a non-linear relationship between input and response is well knownin chemical and physical analysis such as the relationship between graindensity versus photon exposure in chemical film or in a charge-coupleddevice (CCD) where, the relative brightness of photographic inputs aremaintained in relative rank order in the resulting image, yet a detailedunderstanding of the non-linear response is required to predict relativeabundance of the original inputs (analogous to DNA copy number in theoriginal sample) from the response data (analogous to PCR product signalafter amplification and microarray hybridization).

The non-linear relationships described above are similar to thatobserved in chemical and physical analysis, such as the relationshipbetween grain density versus photon exposure in chemical films orcharge-coupled devices (CCD) where, the relative brightness ofphotographic inputs are maintained in relative rank order in theresulting image, yet a detailed understanding of the non-linear responseis required to predict relative abundance of the original inputs(analogous to DNA copy number in the original sample) from the responsedata (analogous to PCR product signal after amplification and microarrayhybridization). In the present invention, it is determined that theobserved competition between unknown microbial DNA and standard DNAdisplay a useful and generally unexpected “X-shaped” relationship, ofthe kind displayed in FIG. 19A.

Analysis of Microbial DNA (rDNA or ITS2) Copy Number by PCR-Microarray.

“Crossover” titration data of the kind shown in FIGS. 19A and 19B weredetermined to be intrinsic to the PCR-Microarray analysis described inthis application. As target microbial DNA copy number is increased, withsynthetic DNA copy number being held constant, the products of themicrobial PCR reaction, and hence signals due to binding of thoseproducts to cognate probes on the microarray surface increase withincreasing microbial DNA copy number, whereas the signals obtained fromthe (fixed) matched internal reference standard decrease in concert(FIGS. 19A-19B). The two curves cross-over at or near copy numberequivalency (arrow, FIG. 19A-19B) where the number of microbial DNAcopies become equal to the number of internal reference standard DNAcopies.

Over the range of PCR conditions consistent with the present invention(i.e. conditions of PCR signals saturating due to competition betweenthe unknown microbial DNA and an added DNA standard) the relationbetween the DNA input and output can be approximated as,C _(n) /C _(o) =P(S _(n) /S _(o))^(x) where,  Equation #1

C_(n)=the number of microbial DNA copies of each type (n) present in theoriginal sample mixture added to the first of two tandem PCR reactionsused to prepare amplicons for microarray analysis,

C_(o)=the number of known synthetic DNA copies (internal referencestandard) added to the first of two PCR reactions used to prepareamplicons for microarray analysis,

S_(n)=relative fluorescence units (RFU) signal data obtained after PCRamplification, and microarray hybridization of the nth microbialspecies, followed by image analysis,

S_(o)=relative fluorescence units (RFU) signal data obtained after PCRamplification, and microarray hybridization of the synthetic DNAspecies, followed by image analysis,

X=a complex exponential factor which defines the functional relationshipbetween the Experimental Microarray Data Ratio (S_(n)/S_(o)) to theunderlying ratio of microbial DNA copies vs synthetic DNA standardcopies present in the original sample (C_(n)/C_(o)).

P=A constant which relates the Experimental Microarray Data ratio(S_(n)/S_(o)) to the concentration of amplified PCR product which bindsto the microarray. In general, for the examples presented here (FIGS.19A and 19B), P=1.

In general X can be a linear function or exponential or relatedfunctional form or a constant which is itself a function of PCRparameters and conditions of microarray analysis and imaging. Based onrepresentative data shown in the present invention, X is approximated asa constant with a value near to 2.

TABLE 22 Experimental Data S_(n)/S_(o) as a function of ExperimentalCopy Number Data C_(n)/C_(o) from the Representative PCR-MicroarrayValidation Studies in FIG. 19A C_(n)/C_(o) Measured DNA Copy NumberRatio in the Original Sample to be (S_(n)/S_(o))² Adjusted analyzed byPCR-Microarray Experimental Microarray S_(n)/S_(o) Measured ExperimentalC_(o) = 3000 copies Data Ratio using X = 2 Microarray Data Ratio 0.010.01 0.1 5,000 RFU/45,000 RFU) 0.1 0.25 0.5 (20,000 RFU/40,000 RFU) 1 11 (32,000 RFU/32,000 RFU) 10 5.3 2.3 (40,000 RFU/15,000 RFU)

In general, the function (X) relates original DNA Copy Number Ratio(C_(n)/C_(o)) to the Experimental Microarray Data Ratio (S_(n)/S_(o))generated from the microarray hybridization RFU signal data. Function(X) can have different functional forms depending on details of the PCRreaction and microarray hybridization. However, it was determined that,if (1) All microbial DNA PCR reactions use the same pair of PCR primersas the internal reference standard; and (2) All hybridization probesapplied to the microarray surface have the same affinity for theircognate DNA sequence produced by the PCR reactions; and (3) All PCRproducts are labeled with the same fluorescent dye for opticaldetection; then, X will approach a constant, which in the data presentedin FIG. 19A assumes a value close to 2. Under these conditions Equation#1 may be simplified asC _(n) =C _(o)(S _(n) /S _(o))²  Equation #2where

C_(n)=The number of microbial DNA copies present in the original sample.

C_(o)=Is adjusted by adding a known number of synthetic DNA standardcopies to the original sample.

X=2 as estimated from experimental data, as in FIG. 19A

P=1 as determined by experiment, as in FIG. 19A

For FIG. 19A, the synthetic DNA reference standard copy number (C_(o))was intentionally held at 3,000 but may be set at any other valueincluding but not limited to 100, 500, 3,000 and 5,000 depending on therange of unknown microbial copies which might be encountered.

In a specific implementation of the present invention for microbialtesting in food or Cannabis or other plant matter or for water testing,State or Federal Regulations might require a specific minimum allowablevalue for microbial contamination. Based on that regulated value andknowledge of the number of rDNA or ITS-2 DNA copies per microbialgenome, the adjustable standard copy number value C_(o) added to theoriginal sample before the PCR and Microarray hybridization steps can beseen as having value as a way to “dial” regulatory standards directlyinto the PCR-Microarray assay.

What is claimed is:
 1. A method for simultaneously quantitating copynumber of DNA for one or more pathogens and a plant in a plant tissuesample, comprising the steps of: a) obtaining a plant tissue samplecomprising one or more pathogens; b) isolating total nucleic acidscomprising DNA and non-DNA nucleic acids from the plant and thepathogens in the plant tissue sample; c) adding a known copy number ofat least one synthetic DNA sequence as an internal reference standard,each of said synthetic DNA sequences comprising: a central region havinga nucleotide sequence distinct from signature sequence determinants inthe pathogen DNA and the plant DNA; and a 5′ end and a 3′ end havingnucleotide sequences substantially identical to a consensus sequence inthe pathogen DNA; d) amplifying, in a first amplification in a singleassay, the pathogen DNA and the at least one synthetic DNA sequence inthe total nucleic acids using at least one first primer pair selectivefor the pathogen DNA and the synthetic DNA and at least one secondprimer pair selective for the plant DNA to generate one or more pathogenDNA-specific first amplicons, plant DNA-specific second amplicons andsynthetic DNA-specific third amplicons; e) amplifying, in a secondamplification, using the one or more pathogen-specific first amplicons,the plant DNA-specific second amplicons and the synthetic DNA-specificthird amplicons as a template and at least one first fluorescent labeledthird primer pair and at least one second fluorescent labeled fourthprimer pair to generate pathogen DNA-specific first fluorescent labeledfourth amplicons, plant DNA-specific second fluorescent labeled fifthamplicons and first fluorescent labeled synthetic DNA-specific sixthamplicons; f) hybridizing the pathogen DNA-specific first fluorescentlabeled fourth amplicons, the plant DNA-specific second fluorescentlabeled fifth amplicons and the first fluorescent labeled syntheticDNA-specific sixth amplicons with nucleic acid probes specific forsignature sequence determinants in the pathogen DNA, in the plant DNAand in the synthetic DNA, respectively, said nucleic acid probesimmobilized at specific known positions on a 3-dimensional latticemicroarray via third fluorescent labeled bifunctional polymer linkers;g) washing the 3-dimensional lattice microarray at least once; h)imaging the 3-dimensional lattice microarray to detect a firstfluorescent signal corresponding to the first fluorescent labeled fourthamplicons or the first fluorescent labeled sixth amplicons, a secondfluorescent signal corresponding to the second fluorescent labeled fifthamplicons and a third fluorescent signal corresponding to the nucleicacid probes immobilized at the specific known positions on the3-dimensional lattice microarray via the third fluorescent labeledbifunctional polymer linkers; i) superimposing the first fluorescentsignal and the second fluorescent signal with the third fluorescentsignal to obtain a superimposed signal image; j) comparing the sequenceof the nucleic acid probe at one or more superimposed signal positionson the microarray with a database of signature sequence determinants fora plurality of pathogen DNAs and plant DNAs thereby identifying thepathogens and the plant in the sample; k) correlating mathematically, afirst fluorescent signal intensity from the pathogen DNA-specific firstfluorescent labeled fourth amplicons with a first fluorescent signalintensity from the synthetic DNA-specific first fluorescent labeledsixth amplicons and the known copy number of the synthetic DNA, therebyquantitating copy number of the pathogen DNA in the sample; and l)correlating mathematically, a second fluorescent signal intensity fromthe plant DNA-specific second fluorescent labeled fifth amplicons with afirst fluorescent signal intensity from the synthetic DNA-specific firstfluorescent labeled sixth amplicons and the known copy number of thesynthetic DNA, thereby quantitating copy number of the plant DNA in thesample.
 2. The method of claim 1, wherein the pathogen is a bacterium, afungus, a virus, a yeast, an algae or a protozoan or a combinationthereof.
 3. The method of claim 1, wherein the pathogen is a bacterium,said consensus sequence comprising the nucleotide sequence of SEQ ID NO:153, the first primer pair comprising the nucleotide sequences of SEQ IDNOS: 1 and 2, or SEQ ID NOS: 3 and 4, or SEQ ID NOS: 5 and 6, or SEQ IDNOS: 7 and 8, or SEQ ID NOS: 9 and 10, or SEQ ID NOS: 11 and 12, or SEQID NOS: 137 and
 138. 4. The method of claim 1, wherein the pathogen is abacterium, said consensus sequence comprising the nucleotide sequence ofSEQ ID NO: 153, the first fluorescent labeled third primer paircomprising the nucleotide sequences of SEQ ID NOS: 19 and 20, or SEQ IDNOS: 21 and 22, or SEQ ID NOS: 23 and 24, or SEQ ID NOS: 25 and 26, orSEQ ID NOS: 27 and 28, or SEQ ID NOS: 29 and 30, or SEQ ID NOS: 141 and30.
 5. The method of claim 1, wherein the pathogen is a bacterium, thenucleic acid probes specific to the bacterium comprising the nucleotidesequences of SEQ ID NOS: 37-85.
 6. The method of claim 1, wherein thepathogen is a bacterium, the synthetic DNA sequence comprising thenucleotide sequences of SEQ ID NO: 155, SEQ ID NO: 156 and SEQ ID NO:157 that hybridize to synthetic DNA sequence specific nucleic acidprobes comprising the nucleotide sequences of SEQ ID NO: 142, SEQ ID NO:143 and SEQ ID NO: 144, respectively.
 7. The method of claim 1, whereinthe pathogen is a fungus, said consensus sequence comprising thenucleotide sequence of SEQ ID NO: 152, the first primer pair comprisingthe nucleotide sequences of SEQ ID NOS: 13 and 14, or SEQ ID NOS: 15 and16, or SEQ ID NOS: 135 and
 136. 8. The method of claim 1, wherein thepathogen is a fungus, said consensus sequence comprising the nucleotidesequence of SEQ ID NO: 152, the first fluorescent labeled third primerpair comprising the nucleotide sequence of SEQ ID NOS: 31 and 32, or SEQID NOS: 33 and 34, or SEQ ID NOS: 139 and
 140. 9. The method of claim 1,wherein the pathogen is a fungus, said nucleic acid probes specific tothe fungus comprising the nucleotide sequences of SEQ ID NOS: 86-125.10. The method of claim 1, wherein the pathogen is a fungus, thesynthetic DNA sequence comprising the nucleotide sequence of SEQ ID NO:154 that hybridizes to synthetic DNA sequence specific nucleic acidprobes comprising the nucleotide sequences of SEQ ID NO:
 145. 11. Themethod of claim 1, wherein the plant is a Humulus or a Cannabis.
 12. Themethod of claim 1, wherein the plant is Cannabis, said second primerpair comprising the nucleotide sequence of SEQ ID NOS: 17 and
 18. 13.The method of claim 1, wherein the plant is Cannabis, said secondfluorescent labeled fourth primer pair comprising the nucleotidesequence of SEQ ID NOS: 35 and
 36. 14. The method of claim 1, whereinthe plant is Cannabis, the nucleic acid probes comprising the nucleotidesequences of SEQ ID NOS: 126-128.
 15. The method of claim 1, wherein thesteps of correlating mathematically is performed using the relationship:C _(n) /C _(o) =P(S _(n) /S _(o))^(x) where, C_(n) is the copy number ofpathogen DNA or plant DNA of each type n of pathogen or plant species tobe quantitated, C_(o) is the known copy number of the synthetic DNAsequence added to the sample prior to performing the first amplificationstep, S_(n) is the first fluorescent signal intensity in relativefluorescence units (RFU) from the pathogen DNA-specific firstfluorescent labeled third amplicons that hybridized to the pathogenDNA-specific nucleic acid probes at the specific known positions on the3-dimensional lattice microarray for each of the type n of pathogenspecies, or the second fluorescent signal intensity in RFU from theplant DNA-specific second fluorescent labeled fourth amplicons thathybridized to the plant DNA-specific nucleic acid probes at the specificknown positions on the microarray for each of the type n plant species,S_(o) is first fluorescent signal intensity in RFU from the syntheticDNA-specific first fluorescent labeled sixth amplicons that hybridizedto the synthetic DNA sequence-specific nucleic acid probes at thespecific known positions on the 3-dimensional lattice microarray, P is aconstant ranging from about 0.1 to about 10 and X is a an exponentialfactor ranging from about 1 to about 3.